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Nutrient availability is a key determinant of tumor cell behavior. While nutrient-rich conditions favor proliferation and tumor growth, scarcity, and particularly glutamine starvation, promotes cell dedifferentiation and chemoresistance. Here, linking ribosome biogenesis plasticity with tumor cell fate, we uncover that the amino acid sensor general control non-derepressible 2 (GCN2; also known as eIF-2-alpha kinase 4) represses the expression of the precursor of ribosomal RNA (rRNA), 47S, under metabolic stress. We show that blockade of GCN2 triggers cell death by an irremediable nucleolar stress and subsequent TP53-mediated apoptosis in patient-derived models of colon adenocarcinoma (COAD). In nutrient-rich conditions, a cell-autonomous GCN2 activity supports cell proliferation by stimulating 47S rRNA transcription, independently of the canonical integrated stress response (ISR) axis. Impairment of GCN2 activity prevents nuclear translocation of methionyl-tRNA synthetase (MetRS), resulting in nucleolar stress, mTORC1 inhibition and, ultimately, autophagy induction. Inhibition of the GCN2-MetRS axis drastically improves the cytotoxicity of RNA polymerase I (RNA pol I) inhibitors, including the first-line chemotherapy oxaliplatin, on patient-derived COAD tumoroids. Our data thus reveal that GCN2 differentially controls ribosome biogenesis according to the nutritional context. Furthermore, pharmacological co-inhibition of the two GCN2 branches and RNA pol I activity may represent a valuable strategy for elimination of proliferative and metabolically stressed COAD cells., (© 2023 The Authors. Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Dormancy in cancer is a clinical state in which residual disease remains undetectable for a prolonged duration. At a cellular level, rare cancer cells cease proliferation and survive chemotherapy and disseminate disease. We created a suspension culture model of high-grade serous ovarian cancer (HGSOC) dormancy and devised a novel CRISPR screening approach to identify survival genes in this context. In combination with RNA-seq, we discovered the Netrin signaling pathway as critical to dormant HGSOC cell survival. We demonstrate that Netrin-1, -3, and its receptors are essential for low level ERK activation to promote survival, and that Netrin activation of ERK is unable to induce proliferation. Deletion of all UNC5 family receptors blocks Netrin signaling in HGSOC cells and compromises viability during the dormancy step of dissemination in xenograft assays. Furthermore, we demonstrate that Netrin-1 and -3 overexpression in HGSOC correlates with poor outcome. Specifically, our experiments reveal that Netrin overexpression elevates cell survival in dormant culture conditions and contributes to greater spread of disease in a xenograft model of abdominal dissemination. This study highlights Netrin signaling as a key mediator HGSOC cancer cell dormancy and metastasis., Competing Interests: PP, JM, KZ, DP, SW, YR, MH, RR, BG, RC, TS, FD No competing interests declared, PM Founder of Netris Pharma, (© 2023, Perampalam et al.)

Nanoparticle's success as drug delivery systems for cancer treatment has been achieved through passive targeting mechanisms. However, tumor heterogeneity and rapid drug clearance limit the treatment efficacy. Improved outcomes and selective drug release can be achieved by grafting ligands at the surface of nanocarriers that bind molecules overexpressed in the tumor microenvironment (TME). In this work, we developed a docetaxel-loaded nanoemulsions (NEs) binding an anti-netrin-1 monoclonal antibody (NP137) to selectively target the netrin-1 protein overexpressed in many different tumors. The goal is to refine a combined approach utilizing NP137 and docetaxel as an improved tumor-targeting chemotherapeutic agent for addressing triple-negative breast cancer (TNBC). Several factors have been considered for the optimization of the active targeted drug delivery system via the click-chemistry conjugation, as the impact of PEGylated surfactant that stabilize the NEs shell on conjugation efficiency, cytocompatibility with EMT6 cell line and colloidal stability over time of NEs. Results showed that a 660 Da PEG chain length contributed to NEs colloidal stability and had no impact on cell viability or on the antibody binding ability for its ligand after surface conjugation. Moreover, docetaxel was encapsulated into the oily core of NEs, with an encapsulation efficiency of 70 %. To validate our treatment strategy in vivo, the 4T1 murine breast cancer model was used. As a result, the comparison of active-targeted and non-targeted NEs revealed that only active-targeted NE could decrease the tumor growth rate., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Giovanna Lollo reports financial support and equipment, drugs, or supplies were provided by University Claude Bernard Lyon 1 - Campus Rockefeller. Benjamin Gibert reports financial support and equipment, drugs, or supplies were provided by Cancer Research Centre Lyon. Ilaria Marigo reports financial support and equipment, drugs, or supplies were provided by Veneto Oncology Institute. Patrick Mehlen declares to have a coflict of interest as shareholder of Netris Pharma. The other authors declare that they have no financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Neuropilin 2 (NRP2), a transmembrane non-tyrosine kinase receptor, has been described as a potential critical player in the tumourigenesis of several solid cancers and particularly in neuroendocrine neoplasms (NENs). A soluble form of NRP2 (sNRP2) has been previously described and corresponds to a truncated splice isoform. Its prognostic value has never been studied in NEN. NRP2 expression was studied by immunochemistry on tissue microarrays (n = 437) and on circulating tumour cells (CTCs, n = 5 patients with neuroendocrine carcinoma, NEC). We described the levels of sNRP2 in 229 patients with NEN using the ELISA method to identify the factors associated with sNRP2 levels and to evaluate its prognostic role; 90 blood donors represented the healthy control group. NRP2 was found in 97% of neuroendocrine tumours (396/410) and in 74% of NEC (20/27). NRP2 was also expressed in CTC of all the studied patients. The receiver operating characteristic (ROC) analysis showed that sNRP2 had a weak capacity to discriminate between NEN patients and healthy controls (area under curve (AUC) = 0.601, P = 0.053). Abnormal sNRP2 levels were associated with inflammatory syndrome, bone and peritoneal metastases, and abnormal chromogranin A levels. Patients with high sNRP2 levels (sNRP2Q3-Q4) had significantly poorer overall survival in multivariate analysis (HR 0.16, 95% CI (0.04-0.67), P = 0.015). In conclusion, the present study found that sNRP2 and NRP2 could represent a new prognostic biomarker and a therapeutic target, respectively, particularly in aggressive NEN.

The nucleus is an essential organelle for the function of cells. It holds most of the genetic material and plays a crucial role in the regulation of cell growth and proliferation. Since many antitumoral therapies target nucleic acids to induce cell death, tumor-specific nuclear drug delivery could potentiate therapeutic effects and prevent potential off-target side effects on healthy tissue. Due to their great structural variety, good biocompatibility, and unique physico-chemical properties, organometallic complexes and other metal-based compounds have sparked great interest as promising anticancer agents. In this review, strategies for specific nuclear delivery of metal complexes are summarized and discussed to highlight crucial parameters to consider for the design of new metal complexes as anticancer drug candidates. Moreover, the existing opportunities and challenges of tumor-specific, nucleus-targeting metal complexes are emphasized to outline some new perspectives and help in the design of new cancer treatments., (© 2024 The Authors. Advanced Materials published by Wiley‐VCH GmbH.)

In breast cancers, aberrant activation of the RAS/MAPK pathway is strongly associated with mesenchymal features and stemness traits, suggesting an interplay between this mitogenic signaling pathway and epithelial-to-mesenchymal plasticity (EMP). By using inducible models of human mammary epithelial cells, we demonstrate herein that the oncogenic activation of RAS promotes ZEB1-dependent EMP, which is necessary for malignant transformation. Notably, EMP is triggered by the secretion of pro-inflammatory cytokines from neighboring RAS-activated senescent cells, with a prominent role for IL-6 and IL-1α. Our data contrast with the common view of cellular senescence as a tumor-suppressive mechanism and EMP as a process promoting late stages of tumor progression in response to signals from the tumor microenvironment. We highlighted here a pro-tumorigenic cooperation of RAS-activated mammary epithelial cells, which leverages on oncogene-induced senescence and EMP to trigger cellular reprogramming and malignant transformation.

Drug resistance and cancer relapse represent significant therapeutic challenges after chemotherapy or immunotherapy, and a major limiting factor for long-term cancer survival. Netrin-1 was initially identified as a neuronal navigation cue but has more recently emerged as an interesting target for cancer therapy, which is currently clinically investigated. We show here that netrin-1 is an independent prognostic marker for clinical progression of breast and ovary cancers. Cancer stem cells (CSCs)/Tumor initiating cells (TICs) are hypothesized to be involved in clinical progression, tumor relapse and resistance. We found a significant correlation between netrin-1 expression and cancer stem cell (CSC) markers levels. We also show in different mice models of resistance to chemotherapies that netrin-1 interference using a therapeutic netrin-1 blocking antibody alleviates resistance to chemotherapy and triggers an efficient delay in tumor relapse and this effect is associated with CSCs loss. We also demonstrate that netrin-1 interference limits tumor resistance to immune checkpoint inhibitor and provide evidence linking this enhanced anti-tumor efficacy to a decreased recruitment of a subtype of myeloid-derived suppressor cells (MDSCs) called polymorphonuclear (PMN)-MDSCs. We have functionally demonstrated that these immune cells promote CSCs features and, consequently, resistance to anti-cancer treatments. Together, these data support the view of both a direct and indirect contribution of netrin-1 to cancer stemness and we propose that this may lead to therapeutic opportunities by combining conventional chemotherapies and immunotherapies with netrin-1 interfering drugs., (© 2023. The Author(s), under exclusive licence to ADMC Associazione Differenziamento e Morte Cellulare.)

Targeted radionuclide therapy is a revolutionary tool for the treatment of highly spread metastatic cancers. Most current approaches rely on the use of vectors to deliver radionuclides to tumor cells, targeting membrane-bound cancer-specific moieties. Here, we report the embryonic navigation cue netrin-1 as an unanticipated target for vectorized radiotherapy. While netrin-1, known to be re-expressed in tumoral cells to promote cancer progression, is usually characterized as a diffusible ligand, we demonstrate here that netrin-1 is actually poorly diffusible and bound to the extracellular matrix. A therapeutic anti-netrin-1 monoclonal antibody (NP137) has been preclinically developed and was tested in various clinical trials showing an excellent safety profile. In order to provide a companion test detecting netrin-1 in solid tumors and allowing the selection of therapy-eligible patients, we used the clinical-grade NP137 agent and developed an indium-111-NODAGA-NP137 single photon emission computed tomography (SPECT) contrast agent. NP137- 111 In provided specific detection of netrin-1-positive tumors with an excellent signal-to-noise ratio using SPECT/CT imaging in different mouse models. The high specificity and strong affinity of NP137 paved the way for the generation of lutetium-177-DOTA-NP137, a novel vectorized radiotherapy, which specifically accumulated in netrin-1-positive tumors. We demonstrate here, using tumor cell-engrafted mouse models and a genetically engineered mouse model, that a single systemic injection of NP137- 177 Lu provides important antitumor effects and prolonged mouse survival. Together, these data support the view that NP137- 111 In and NP137- 177 Lu may represent original and unexplored imaging and therapeutic tools against advanced solid cancers., (© 2023 The Authors. Published under the terms of the CC BY 4.0 license.)

Background and Aims: Netrin-1 displays protumoral properties, though the pathological contexts and processes involved in its induction remain understudied. The liver is a major model of inflammation-associated cancer development, leading to HCC., Approach and Results: A panel of cell biology and biochemistry approaches (reverse transcription quantitative polymerase chain reaction, reporter assays, run-on, polysome fractionation, cross linking immunoprecipitation, filter binding assay, subcellular fractionation, western blotting, immunoprecipitation, stable isotope labeling by amino acids in cell culture) on in vitro-grown primary hepatocytes, human liver cell lines, mouse samples and clinical samples was used. We identify netrin-1 as a hepatic inflammation-inducible factor and decipher its mode of activation through an exhaustive eliminative approach. We show that netrin-1 up-regulation relies on a hitherto unknown mode of induction, namely its exclusive translational activation. This process includes the transfer of NTN1 (netrin-1) mRNA to the endoplasmic reticulum and the direct interaction between the Staufen-1 protein and this transcript as well as netrin-1 mobilization from its cell-bound form. Finally, we explore the impact of a phase 2 clinical trial-tested humanized anti-netrin-1 antibody (NP137) in two distinct, toll-like receptor (TLR) 2/TLR3/TLR6-dependent, hepatic inflammatory mouse settings. We observe a clear anti-inflammatory activity indicating the proinflammatory impact of netrin-1 on several chemokines and Ly6C+ macrophages., Conclusions: These results identify netrin-1 as an inflammation-inducible factor in the liver through an atypical mechanism as well as its contribution to hepatic inflammation., (© 2022 American Association for the Study of Liver Diseases.)

Since the Nobel Prize award more than twenty years ago for discovering the core apoptotic pathway in C. elegans , apoptosis and various other forms of regulated cell death have been thoroughly characterized by researchers around the world. Although many aspects of regulated cell death still remain to be elucidated in specific cell subtypes and disease conditions, many predicted that research into cell death was inexorably reaching a plateau. However, this was not the case since the last decade saw a multitude of cell death modalities being described, while harnessing their therapeutic potential reached clinical use in certain cases. In line with keeping research into cell death alive, francophone researchers from several institutions in France and Belgium established the French Cell Death Research Network (FCDRN). The research conducted by FCDRN is at the leading edge of emerging topics such as non-apoptotic functions of apoptotic effectors, paracrine effects of cell death, novel canonical and non-canonical mechanisms to induce apoptosis in cell death-resistant cancer cells or regulated forms of necrosis and the associated immunogenic response. Collectively, these various lines of research all emerged from the study of apoptosis and in the next few years will increase the mechanistic knowledge into regulated cell death and how to harness it for therapy.

Background: Before the advent of an effective vaccine, nonpharmaceutical interventions, such as mask-wearing, social distancing, and lockdowns, have been the primary measures to combat the COVID-19 pandemic. Such measures are highly effective when there is high population-wide adherence, which requires information on current risks posed by the pandemic alongside a clear exposition of the rules and guidelines in place., Objective: Here we analyzed online news media coverage of COVID-19. We quantified the total volume of COVID-19 articles, their sentiment polarization, and leading subtopics to act as a reference to inform future communication strategies., Methods: We collected 26 million news articles from the front pages of 172 major online news sources in 11 countries (available online at SciRide). Using topic detection, we identified COVID-19-related content to quantify the proportion of total coverage the pandemic received in 2020. The sentiment analysis tool Vader was employed to stratify the emotional polarity of COVID-19 reporting. Further topic detection and sentiment analysis was performed on COVID-19 coverage to reveal the leading themes in pandemic reporting and their respective emotional polarizations., Results: We found that COVID-19 coverage accounted for approximately 25.3% of all front-page online news articles between January and October 2020. Sentiment analysis of English-language sources revealed that overall COVID-19 coverage was not exclusively negatively polarized, suggesting wide heterogeneous reporting of the pandemic. Within this heterogenous coverage, 16% of COVID-19 news articles (or 4% of all English-language articles) can be classified as highly negatively polarized, citing issues such as death, fear, or crisis., Conclusions: The goal of COVID-19 public health communication is to increase understanding of distancing rules and to maximize the impact of governmental policy. The extent to which the quantity and quality of information from different communication channels (eg, social media, government pages, and news) influence public understanding of public health measures remains to be established. Here we conclude that a quarter of all reporting in 2020 covered COVID-19, which is indicative of information overload. In this capacity, our data and analysis form a quantitative basis for informing health communication strategies along traditional news media channels to minimize the risks of COVID-19 while vaccination is rolled out., (©Konrad Krawczyk, Tadeusz Chelkowski, Daniel J Laydon, Swapnil Mishra, Denise Xifara, Seth Flaxman, Seth Flaxman, Thomas Mellan, Veit Schwämmle, Richard Röttger, Johannes T Hadsund, Samir Bhatt. Originally published in the Journal of Medical Internet Research (https://www.jmir.org), 02.06.2021.)

The navigation cue netrin-1 is well-documented for its key role in cancer development and represents a promising therapeutic target currently under clinical investigation. Phase 1 and 2 clinical trials are ongoing with NP137, a humanized monoclonal antibody against netrin-1. Interestingly, the epitope recognized by NP137 in netrin-1 shares 90% homology with its counterpart in netrin-3, the closest member to netrin-1 in humans, for which little is known in the field of cancer. Here, we unveiled that netrin-3 appears to be expressed specifically in human neuroblastoma (NB) and small cell lung cancer (SCLC), two subtypes of neuroectodermal/neuroendocrine lineages. Netrin-3 and netrin-1 expression are mutually exclusive, and the former is driven by the MYCN oncogene in NB, and the ASCL-1 or NeuroD1 transcription factors in SCLC. Netrin-3 expression is correlated with disease stage, aggressiveness, and overall survival in NB. Mechanistically, we confirmed the high affinity of netrin-3 for netrin-1 receptors and we demonstrated that netrin-3 genetic silencing or interference using NP137, delayed tumor engraftment, and reduced tumor growth in animal models. Altogether, these data support the targeting of netrin-3 in NB and SCLC., (© 2021 The Authors. Published under the terms of the CC BY 4.0 license.)

Haematopoietic stem cells (HSCs) are characterized by their self-renewal potential associated to dormancy. Here we identify the cell surface receptor neogenin-1 as specifically expressed in dormant HSCs. Loss of neogenin-1 initially leads to increased HSC expansion but subsequently to loss of self-renewal and premature exhaustion in vivo. Its ligand netrin-1 induces Egr1 expression and maintains quiescence and function of cultured HSCs in a Neo1 dependent manner. Produced by arteriolar endothelial and periarteriolar stromal cells, conditional netrin-1 deletion in the bone marrow niche reduces HSC numbers, quiescence and self-renewal, while overexpression increases quiescence in vivo. Ageing associated bone marrow remodelling leads to the decline of netrin-1 expression in niches and a compensatory but reversible upregulation of neogenin-1 on HSCs. Our study suggests that niche produced netrin-1 preserves HSC quiescence and self-renewal via neogenin-1 function. Decline of netrin-1 production during ageing leads to the gradual decrease of Neo1 mediated HSC self-renewal.

Introduction: Gastroenteropancreatic neuroendocrine carcinomas (GEPNEC) are characterized by a heterogeneous molecular profile and a poor prognosis. Circulating tumour DNA (ctDNA) analysis may be useful for NEC management. This study aimed at describing ctDNA mutations, to assess their predictive value for response to chemotherapies, and their change according to disease progression., Methods: The CIRCAN-NEC study included patients with GEPNEC or NEC from an unknown primary, scheduled to begin first- or second-line chemotherapy. Blood samples were collected prior to chemotherapy initiation, at first evaluation, and during disease progression. ctDNA was sequenced by next-generation sequencing (NGS). Molecular response was defined as a decrease of at least 30% of the mutant allele fraction., Results: All 24 patients included received platinum-etoposide first-line chemotherapy; 19 received a FOLFIRI-based post-first-line regimen. Twenty-two patients had at least one driver mutation: TP53 (n = 21), RB1 (n = 2), KRAS (n = 4), and BRAF (n = 3). Ten (42%) had an "adenocarcinoma-like" profile. Five of 6 patients with matching ctDNA/tissue NGS harboured at least one concordant mutation (44% concordance at the gene level). The concordance rate between ctDNA mutation/immunohistochemistry profile was 64% (7/11) for TP53/p53+ and 14% (1/7) for RB1/pRb-. In this pilot study including few patients by subgroups, patients with KRAS (HR = 3.60, 95% CI [1.06-12.04]) and BRAF (HR = 4.25, 95% CI [1.11-16.40]) mutations had shorter progression-free survival (PFS) under platinum-etoposide, while the 2 patients with RB1 mutations had shorter PFS under FOLFIRI-based chemotherapy. Twenty-eight periods of treatment were assessed: 10 patients had a molecular response (7/10 had a morphological response), which was associated with longer PFS (HR = 0.37, 95% CI [0.15; 0.91])., Conclusion: This pilot study shows a high sensitivity of ctDNA assessment, which is encouraging for the future management of GEPNEC (tumour molecular diagnosis and evaluation of disease progression)., (© 2020 S. Karger AG, Basel.)

Introduction: Small-intestinal neuroendocrine tumors (SI-NET) are situated preferentially within the ileum. The aim was to describe a potential difference in location between unifocal and multiple ileal-NET., Patients and Methods: Between December 2010 and December 2019, all consecutive patients who underwent resection in our European Neuroendocrine Tumor Society Center of Excellence, of at least 1 non-duodenal SI-NET, were retrospectively included. The main objective was to prove that multiple ileal-NET were mostly located on the left side of the superior mesenteric artery (SMA) axis (defined as 40 cm from the ileocecal valve), and unifocal ones on the right side., Results: Ninety-four patients were included, 6 with unifocal jejunal-NET located 35 cm (range, 10-60) from the duodenojejunal angle (DJA), 44 (47%) with unifocal ileal-NET and 44 (47%) with multiple ileal-NET. The median number of tumors in multiple ileal-NET was 7 (range, 2-95), within a median small bowel segment of 105 cm (10-240). The median length between the proximal tumor and the DJA was 428 cm (300-635) and 540 cm (350-725) for the distal one; 40 (91%) of them were located on the left side of the SMA axis. In contrast, unifocal ileal-NET were located at a median distance of 577 cm (305-820) from the DJA (p < 0.001, compared to multiple ileal-NET); 30 (68%) of them were on the right side of the SMA axis (p < 0.001)., Conclusion: Multiple ileal-NET are mostly located on the left side of the SMA axis. Further studies are warranted to explore the embryological origin of unifocal versus multiple ileal-NET., (© 2020 S. Karger AG, Basel.)

Epigenetic deregulation of gene transcription is central to cancer cell plasticity and malignant progression but remains poorly understood. We found that the uncharacterized epigenetic factor chromodomain on Y-like 2 (CDYL2) is commonly over-expressed in breast cancer, and that high CDYL2 levels correlate with poor prognosis. Supporting a functional role for CDYL2 in malignancy, it positively regulated breast cancer cell migration, invasion, stem-like phenotypes, and epithelial-to-mesenchymal transition. CDYL2 regulation of these plasticity-associated processes depended on signaling via p65/NF-κB and STAT3. This, in turn, was downstream of CDYL2 regulation of MIR124 gene transcription. CDYL2 co-immunoprecipitated with G9a/EHMT2 and GLP/EHMT1 and regulated the chromatin enrichment of G9a and EZH2 at MIR124 genes. We propose that CDYL2 contributes to poor prognosis in breast cancer by recruiting G9a and EZH2 to epigenetically repress MIR124 genes, thereby promoting NF-κB and STAT3 signaling, as well as downstream cancer cell plasticity and malignant progression., Competing Interests: Declaration of Interests The authors have no conflicts of interest to declare., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Triggering apoptosis remains an efficient strategy to treat cancer. However, apoptosis is no longer a final destination since cancer cells can undergo partial apoptosis without dying. Recent evidence shows that partial mitochondrial permeabilization and non-lethal caspase activation occur under certain circumstances, although it remains unclear how failed apoptosis affects cancer cells. Using a cancer cell model to trigger non-lethal caspase activation, we find that melanoma cancer cells undergoing failed apoptosis have a particular transcriptomic signature associated with focal adhesions, transendothelial migration, and modifications of the actin cytoskeleton. In line with this, cancer cells surviving apoptosis gain migration and invasion properties in vitro and in vivo. We further demonstrate that failed apoptosis-associated gain in invasiveness is regulated by the c-Jun N-terminal kinase (JNK) pathway, whereas its RNA sequencing signature is found in metastatic melanoma. These findings advance our understanding of how cell death can both cure and promote cancer., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

In ultrasound molecular imaging (USMI), ligand-functionalized microbubbles (MBs) are used to visualize vascular endothelial targets. Netrin-1 is upregulated in 60% of metastatic breast cancers and promotes tumor progression. A novel netrin-1 interference therapy requires the assessment of netrin-1 expression prior to treatment. In this study, we studied netrin-1 as a target for USMI and its potential as a companion diagnostic in breast cancer models. Methods: To verify netrin-1 expression and localization, an in vivo immuno-localization approach was applied, in which anti-netrin-1 antibody was injected into living mice 24 h before tumor collection, and revealed with secondary fluorescent antibody for immunofluorescence analysis. Netrin-1 interactions with the cell surface were studied by flow cytometry. Netrin-1-targeted MBs were prepared using MicroMarker Target-Ready (VisualSonics), and validated in in vitro binding assays in static conditions or in a flow chamber using purified netrin-1 protein or netrin-1-expressing cancer cells. In vivo USMI of netrin-1 was validated in nude mice bearing human netrin-1-positive SKBR7 tumors or weakly netrin-1-expressing MDA-MB-231 tumors using the Vevo 2100 small animal imaging device (VisualSonics). USMI feasibility was further tested in transgenic murine FVB/N Tg(MMTV/PyMT634Mul) (MMTV-PyMT) mammary tumors. Results: Netrin-1 co-localized with endothelial CD31 in netrin-1-positive breast tumors. Netrin-1 binding to the surface of endothelial HUVEC and cancer cells was partially mediated by heparan sulfate proteoglycans. MBs targeted with humanized monoclonal anti-netrin-1 antibody bound to netrin-1-expressing cancer cells in static and dynamic conditions. USMI signal was significantly increased with anti-netrin-1 MBs in human SKBR7 breast tumors and transgenic murine MMTV-PyMT mammary tumors compared to signals recorded with either isotype control MBs or after blocking of netrin-1 with humanized monoclonal anti-netrin-1 antibody. In weakly netrin-1-expressing human tumors and normal mammary glands, no difference in imaging signal was observed with anti-netrin-1- and isotype control MBs. Ex vivo analysis confirmed netrin-1 expression in MMTV-PyMT tumors. Conclusions: These results show that USMI allowed reliable detection of netrin-1 on the endothelium of netrin-1-positive human and murine tumors. Significant differences in USMI signal for netrin-1 reflected the significant differences in netrin-1 mRNA & protein expression observed between different breast tumor models. The imaging approach was non-invasive and safe, and provided the netrin-1 expression status in near real-time. Thus, USMI of netrin-1 has the potential to become a companion diagnostic for the stratification of patients for netrin-1 interference therapy in future clinical trials., Competing Interests: Competing Interests: D. Goldschneider is an employee of Netris Pharma. J.G. Delcros is the inventor of the anti-netrin-1 antibody (patent EP2893939A1). J.G. Delcros and B. Gibert are consultants for Netris Pharma. P. Mehlen is a shareholder of Netris Pharma. The authors J. Wischhusen, K. E. Wilson, R. Molina-Peña, S. Jiang, J. Ngo, and J. K. Willmann, and F. Padilla declare no potential conflicts of interest.

The importance of regulated necrosis in pathologies such as cerebral stroke and myocardial infarction is now fully recognized. However, the physiological relevance of regulated necrosis remains unclear. Here, we report a conserved role for p53 in regulating necrosis in Drosophila and mammalian spermatogenesis. We found that Drosophila p53 is required for the programmed necrosis that occurs spontaneously in mitotic germ cells during spermatogenesis. This form of necrosis involved an atypical function of the initiator caspase Dronc/Caspase 9, independent of its catalytic activity. Prevention of p53-dependent necrosis resulted in testicular hyperplasia, which was reversed by restoring necrosis in spermatogonia. In mouse testes, p53 was required for heat-induced germ cell necrosis, indicating that regulation of necrosis is a primordial function of p53 conserved from invertebrates to vertebrates. Drosophila and mouse spermatogenesis will thus be useful models to identify inducers of necrosis to treat cancers that are refractory to apoptosis.

Notch signalling is a causal determinant of cancer and efforts have been made to develop targeted therapies to inhibit the so-called canonical pathway. Here we describe an unexpected pro-apoptotic role of Notch3 in regulating tumour angiogenesis independently of the Notch canonical pathway. The Notch3 ligand Jagged-1 is upregulated in a fraction of human cancer and our data support the view that Jagged-1, produced by cancer cells, is inhibiting the apoptosis induced by the aberrant Notch3 expression in tumour vasculature. We thus present Notch3 as a dependence receptor inducing endothelial cell death while this pro-apoptotic activity is blocked by Jagged-1. Along this line, using Notch3 mutant mice, we demonstrate that tumour growth and angiogenesis are increased when Notch3 is silenced in the stroma. Consequently, we show that the well-documented anti-tumour effect mediated by γ-secretase inhibition is at least in part dependent on the apoptosis triggered by Notch3 in endothelial cells.

Netrins, a family of laminin-related molecules, have been proposed to act as guidance cues either during nervous system development or the establishment of the vascular system. This was clearly demonstrated for netrin-1 via its interaction with the receptors DCC and UNC5s. However, mainly based on shared homologies with netrin-1, netrin-4 was also proposed to play a role in neuronal outgrowth and developmental/pathological angiogenesis via interactions with netrin-1 receptors. Here, we present the high-resolution structure of netrin-4, which shows unique features in comparison with netrin-1, and show that it does not bind directly to any of the known netrin-1 receptors. We show that netrin-4 disrupts laminin networks and basement membranes (BMs) through high-affinity binding to the laminin γ1 chain. We hypothesize that this laminin-related function is essential for the previously described effects on axon growth promotion and angiogenesis. Our study unveils netrin-4 as a non-enzymatic extracellular matrix protein actively disrupting pre-existing BMs.

In a number of human cancers, NTN1 upregulation inhibits apoptosis induced by its so-called dependence receptors DCC and UNC5H, thus promoting tumor progression. In other cancers however, the selective inhibition of this dependence receptor death pathway relies on the silencing of pro-apoptotic effector proteins. We show here that a substantial fraction of human breast tumors exhibits simultaneous DNA methylation-dependent loss of expression of NTN1 and of DAPK1, a serine threonine kinase known to transduce the netrin-1 dependence receptor pro-apoptotic pathway. The inhibition of DNA methylation by drugs such as decitabine restores the expression of both NTN1 and DAPK1 in netrin-1-low cancer cells. Furthermore, a combination of decitabine with NTN1 silencing strategies or with an anti-netrin-1 neutralizing antibody potentiates tumor cell death and efficiently blocks tumor growth in different animal models. Thus, combining DNA methylation inhibitors with netrin-1 neutralizing agents may be a valuable strategy for combating cancer., (© 2016 The Authors. Published under the terms of the CC BY 4.0 license.)

Data accumulating over the last 20 years support the notion that some transmembrane receptors are activated not only by their respective ligands but also, differentially, by the withdrawal or absence of these same ligands. In this latter setting, these receptors actively trigger apoptosis. They have been dubbed dependence receptors because their expression confers a state of ligand dependence for survival on the expressing cells. Twenty of these receptors have been identified to date, and several have been shown to inhibit tumor progression by inducing apoptosis. As a corollary, these receptors, or their transduced death signals, are frequently silenced in cancer cells as a selective mechanism to prevent cell death, allowing invasion and metastasis. Drugs aimed at inducing programmed cell death in neoplastic cells by re-engaging the proapoptotic activity induced by unliganded dependence receptors are in late-stage preclinical tests, poised for clinical evaluation. This approach may offer novel opportunities for patient treatments. In this review, we discuss the implications of dependence receptors in limiting cancer progression and address the therapeutic perspectives brought to light by this paradigm., (©2015 American Association for Cancer Research.)

The generation of induced pluripotent stem (iPS) cells holds great promise in regenerative medicine. The use of the transcription factors Oct4, Sox2, Klf4 and c-Myc for reprogramming is extensively documented, but comparatively little is known about soluble molecules promoting reprogramming. Here we identify the secreted cue Netrin-1 and its receptor DCC, described for their respective survival/death functions in normal and oncogenic contexts, as reprogramming modulators. In various somatic cells, we found that reprogramming is accompanied by a transient transcriptional repression of Netrin-1 mediated by an Mbd3/Mta1/Chd4-containing NuRD complex. Mechanistically, Netrin-1 imbalance induces apoptosis mediated by the receptor DCC in a p53-independent manner. Correction of the Netrin-1/DCC equilibrium constrains apoptosis and improves reprogramming efficiency. Our work also sheds light on Netrin-1's function in protecting embryonic stem cells from apoptosis mediated by its receptor UNC5b, and shows that the treatment with recombinant Netrin-1 improves the generation of mouse and human iPS cells.

Background: The Sonic Hedgehog (SHH) signaling pathway plays an important role in neural crest cell fate during embryonic development and has been implicated in the progression of multiple cancers that include neuroblastoma, a neural crest cell-derived disease. While most of the SHH signaling is mediated by the well-described canonical pathway leading to the activation of Smoothened and Gli, it has recently been shown that cell-adhesion molecule-related/downregulated by oncogenes (CDON) serves as a receptor for SHH and contributes to SHH-induced signaling. CDON has also been recently described as a dependence receptor, triggering apoptosis in the absence of SHH. This CDON proapoptotic activity has been suggested to constrain tumor progression., Methods: CDON expression was analyzed by quantitative-reverse transcription-polymerase chain reaction in a panel of 226 neuroblastoma patients and associated with stages, overall survival, and expression of miR181 family members using Kaplan Meier and Pearson correlation methods. Cell death assays were performed in neuroblastoma cell lines and tumor growth was investigated in the chick chorioallantoic model. All statistical tests were two-sided., Results: CDON expression was inversely associated with neuroblastoma aggressiveness (P < .001). Moreover, re-expression of CDON in neuroblastoma cell lines was associated with apoptosis in vitro and tumor growth inhibition in vivo. We show that CDON expression is regulated by the miR181 miRNA family, whose expression is directly associated with neuroblastoma aggressiveness (survival: high miR181-b 73.2% vs low miR181-b 54.6%; P = .03)., Conclusions: Together, these data support the view that CDON acts as a tumor suppressor in neuroblastomas, and that CDON is tightly regulated by miRNAs., (© The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Human small heat shock proteins are molecular chaperones that regulate fundamental cellular processes in normal unstressed cells as well as in many cancer cells where they are over-expressed. These proteins are characterized by cell physiology dependent changes in their oligomerization and phosphorylation status. These structural changes allow them to interact with many different client proteins that subsequently display modified activity and/or half-life. Nowdays, the protein interactomes of small Hsps are under intense investigations and will represent, when completed, key parameters to elaborate therapeutic strategies aimed at modulating the functions of these chaperones. Here, we have analyzed the potential pro-cancerous roles of several client proteins that have been described so far to interact with HspB1 (Hsp27) and its close members HspB5 (αB-crystallin) and HspB4 (αA-crystallin).

The secreted factor netrin-1 is upregulated in a fraction of human cancers as a mechanism to block apoptosis induced by netrin-1 dependence receptors DCC and UNC5H. Targeted therapies aiming to trigger tumour cell death via netrin-1/receptors interaction interference are under preclinical evaluation. We show here that Doxorubicin, 5-Fluorouracil, Paclitaxel and Cisplatin treatments trigger, in various human cancer cell lines, an increase of netrin-1 expression which is accompanied by netrin-1 receptors increase. This netrin-1 upregulation which appears to be p53-dependent is a survival mechanism as netrin-1 silencing by siRNA is associated with a potentiation of cancer cell death upon Doxorubicin treatment. We show that candidate drugs interfering with netrin-1/netrin-1 receptors interactions potentiate Doxorubicin, Cisplatin or 5-Fluorouracil-induced cancer cell death in vitro. Moreover, in a model of xenografted nude mice, we show that systemic Doxorubicin treatment triggers netrin-1 upregulation in the tumour but not in normal organs, enhancing and prolonging tumour growth inhibiting effect of a netrin-1 interfering drug. Together these data suggest that combining conventional chemotherapies with netrin-1 interference could be a promising therapeutic approach., (© 2013 The Authors. Published by John Wiley and Sons, Ltd on behalf of EMBO.)

The semaphorin guidance molecules and their receptors, the plexins, are often inappropriately expressed in cancers. However, the signaling processes mediated by plexins in tumor cells are still poorly understood. Here, we demonstrate that the Semaphorin 3E (Sema3E) regulates tumor cell survival by suppressing an apoptotic pathway triggered by the Plexin D1 dependence receptor. In mouse models of breast cancer, a ligand trap that sequesters Sema3E inhibited tumor growth and reduced metastasis through a selective tumor cytocidal effect. We further showed that Plexin D1 triggers apoptosis via interaction with the orphan nuclear receptor NR4A1. These results define a critical role of Sema3E/Plexin D1 interaction in tumor resistance to apoptosis and suggest a therapeutic approach based on activation of a dependence receptor pathway., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Several human small heat shock proteins (sHsps) are phosphorylated oligomeric chaperones that enhance stress resistance. They are characterized by their ability to interact and form polydispersed hetero-oligomeric complexes. We have analyzed the cellular consequences of the stable expression of either wild type HspB5 or its cataracts and myopathies inducing R120G mutant in growing and oxidative stress treated HeLa cells that originally express only HspB1. Here, we describe that wild type and mutant HspB5 induce drastic and opposite effects on cell morphology and oxidative stress resistance. The cellular distribution and phosphorylation of these polypeptides as well as the oligomerization profile of the resulting hetero-oligomeric complexes formed by HspB1 with the two types of exogenous polypeptides revealed the dominant effects induced by HspB5 polypeptides towards HspB1. The R120G mutation enhanced the native size and salt resistance of HspB1-HspB5 complex. However, in oxidative conditions the interaction between HspB1 and mutant HspB5 was drastically modified resulting in the aggregation of both partners. The mutation also induced the redistribution of HspB1 phosphorylated at serine 15, originally observed at the level of the small oligomers that do not interact with wild type HspB5, to the large oligomeric complex formed with mutant HspB5. This phosphorylation stabilized the interaction of HspB1 with mutant HspB5. A dominant negative effect towards HspB1 appears therefore as an important event in the cellular sensitivity to oxidative stress mediated by mutated HspB5 expression. These observations provide novel data that describe how a mutated sHsp can alter the protective activity of another member of this family of chaperones.

Purpose: The recent discoveries in the field of human small heat shock proteins (sHSPs) clearly point to the important roles played by these adenosine triphosphate (ATP)-independent chaperones in the regulation of a large spectrum of vital cellular processes and in pathological diseases. These proteins are therefore considered as very attractive therapeutic targets., Aims: To understand the functions of the stress-inducible members of the sHSP family, HspB1, HspB5 and HspB8, and be able to therapeutically modulate their activities, researchers are faced with the complex oligomerisation and phosphorylation properties of these proteins and with their ability to interact with each other and with specific protein targets. Here, we have integrated, in a functionally orientated way, the up-to-date literature data concerning HspB1, HspB5 and HspB8 protein interactions which reflect their numerous crucial cellular functions. We also present data supporting the idea that specific phospho-oligomeric domains of HspB1 are involved in the interaction with particular client proteins., Conclusions: More information concerning the interactions between client protein targets and sHSPs or the multiple combinatorial chimeric oligomeric complexes formed by different sHSPs are urgently required to elaborate a comprehensive sHSPs protein interactome and propose efficient and pathology-specific therapeutic approaches.

Human HSP27 (HSPB1) is a molecular chaperone sensor which, through dynamic changes in its phosphorylation and oligomerization, allows cells to adapt to changes in their physiology and/or mount a protective response to injuries. In pathological conditions, the high level of HSPB1 expression can either be beneficial, such as in diseases characterized by cellular degenerations, or be malignant in cancer cells where it promotes tumourigenesis, metastasis and anti-cancer drug resistance. Structural changes allow HSPB1 to interact with specific client protein partners in order to modulate their folding/activity and/or half-life. Therefore, the search is open for therapeutic compounds aimed at either down- or upregulating HSPB1 activity. In this respect, we have previously described two peptide aptamers (PA11 and PA50) that specifically interact with HSPB1 small oligomers and decrease its anti-apoptotic and tumourigenic activities. A novel analysis of the different HSPB1-interacting aptamers that were isolated earlier revealed that one aptamer (PA23) has the intriguing ability to stimulate the protective activity of HSPB1. We show here that this aptamer abolishes the dominant negative effect induced by the R120G mutant of αB-crystallin (HSPB5) by disrupting its interaction with HSPB1. Hence, developing structure-based interfering strategies could lead to the discovery of HSPB1-based therapeutic drugs.

The Hedgehog signaling is a determinant pathway for tumor progression. However, while inhibition of the Hedgehog canonical pathway-Patched-Smoothened-Gli-has proved efficient in human tumors with activating mutations in this pathway, recent clinical data have failed to show any benefit in other cancers, even though Sonic Hedgehog (SHH) expression is detected in these cancers. Cell-adhesion molecule-related/down-regulated by Oncogenes (CDON), a positive regulator of skeletal muscle development, was recently identified as a receptor for SHH. We show here that CDON behaves as a SHH dependence receptor: it actively triggers apoptosis in the absence of SHH. The pro-apoptotic activity of unbound CDON requires a proteolytic cleavage in its intracellular domain, allowing the recruitment and activation of caspase-9. We show that by inducing apoptosis in settings of SHH limitation, CDON expression constrains tumor progression, and as such, decreased CDON expression observed in a large fraction of human colorectal cancer is associated in mice with intestinal tumor progression. Reciprocally, we propose that the SHH expression, detected in human cancers and previously considered as a mechanism for activation of the canonical pathway in an autocrine or paracrine manner, actually provides a selective tumor growth advantage by blocking CDON-induced apoptosis. In support of this notion, we present the preclinical demonstration that interference with the SHH-CDON interaction triggers a CDON-dependent apoptosis in vitro and tumor growth inhibition in vivo. The latter observation qualifies CDON as a relevant alternative target for anticancer therapy in SHH-expressing tumors., Competing Interests: Agnes Bernet and Patrick Mehlen are co-founders of Netris Pharma.

Hsp27 belongs to the heat shock protein family and displays chaperone properties in stress conditions by holding unfolded polypeptides, hence avoiding their inclination to aggregate. Hsp27 is often referenced as an anti-cancer therapeutic target, but apart from its well-described ability to interfere with different stresses and apoptotic processes, its role in non-stressed conditions is still not well defined. In the present study we report that three polypeptides (histone deacetylase HDAC6, transcription factor STAT2 and procaspase-3) were degraded in human cancerous cells displaying genetically decreased levels of Hsp27. In addition, these proteins interacted with Hsp27 complexes of different native size. Altogether, these findings suggest that HDAC6, STAT2 and procaspase-3 are client proteins of Hsp27. Hence, in non stressed cancerous cells, the structural organization of Hsp27 appears to be a key parameter in the regulation by this chaperone of the level of specific polypeptides through client-chaperone type of interactions.

Human HspB1 (also denoted Hsp27) is an oligomeric anti-apoptotic protein that has tumorigenic and metastatic roles. To approach the structural organizations of HspB1 that are active in response to apoptosis inducers acting through different pathways, we have analyzed the relative protective efficiency induced by this protein as well its localization, oligomerization and phosphorylation. HeLa cells, that constitutively express high levels of HspB1 were treated with either etoposide, Fas agonist antibody, staurosporine or cytochalasin D. Variability in HspB1 efficiency to interfere with the different apoptotic transduction pathways induced by these agents were detected. Moreover, inducer-specific dynamic changes in HspB1 localization, native size and phosphorylation were observed, that differed from those observed after heat shock. Etoposide and Fas treatments gradually shifted HspB1 towards large but differently phosphorylated oligomeric structures. In contrast, staurosporine and cytochalasin D induced the rapid but transient formation of small oligomers before large structures were formed. These events correlated with inducer-specific phosphorylations of HspB1. Of interest, the formation of small oligomers in response to staurosporine and cytochalasin D was time correlated with the rapid disruption of F-actin. The subsequent, or gradual in the case of etoposide and Fas, formation of large oligomeric structures was a later event concomitant with the early phase of caspase activation. These observations support the hypothesis that HspB1 has the ability, through specific changes in its structural organization, to adapt and interfere at several levels with challenges triggered by different signal transduction pathways upstream of the execution phase of apoptosis., (Copyright 2010 Elsevier Inc. All rights reserved.)

Hsp27 and alphaB-crystallin are molecular chaperones that are constitutively expressed in several mammalian cells, particularly in pathological conditions. These proteins share functions as diverse as protection against toxicity mediated by aberrantly folded proteins or oxidative-inflammation conditions. In addition, these proteins share anti-apoptotic properties and are tumorigenic when expressed in cancer cells. This review summarizes the current knowledge about Hsp27 and alphaB-crystallin and the implications, either positive or deleterious, of these proteins in pathologies such as neurodegenerative diseases, myopathies, asthma, cataracts and cancers. Approaches towards therapeutic strategies aimed at modulating the expression and/or the activities of Hsp27 and alphaB-crystallin are presented.