Yves Lepelletier

Neuropilin-1 is not a marker of human Foxp3+ Treg

Treg are immune cells that play a critical role in the regulation of the immune response. Although the transcription factor Foxp3 is widely accepted as the standard marker of Treg, specific surface markers are needed to better characterize these cells and decipher their mechanisms of action. Neuropilin‐1 (Nrp‐1), a membrane protein primarily involved in the nervous system, was identified as a specific marker of murine Treg, but its expression has not been rigorously investigated in human Treg. Here we show that in contrast to murine Treg and regardless of their origins (blood, thymus, spleen, lymph node or tonsil), human Foxp3+ Treg do not specifically express Nrp‐1. However, a population of Foxp3− Nrp‐1+ T cells can be detected in human secondary lymphoid organs, and Nrp‐1 expression is induced on peripheral blood T lymphocytes upon in vitro activation. We conclude that Nrp‐1 cannot be used as a specific marker of human Treg, but might represent a novel activation marker of human T cells both in vitro and in vivo.

p120-Ras GTPase activating protein (RasGAP): a multi-interacting protein in downstream signaling.

p120-RasGAP (Ras GTPase activating protein) plays a key role in the regulation of Ras-GTP bound by promoting GTP hydrolysis via its C-terminal catalytic domain. The p120-RasGAP N-terminal part contains two SH2, SH3, PH (pleckstrin homology) and CaLB/C2 (calcium-dependent phospholipid-binding domain) domains. These protein domains allow various functions, such as anti-/pro-apoptosis, proliferation and also cell migration depending of their distinct partners. The p120-RasGAP domain participates in protein-protein interactions with Akt, Aurora or RhoGAP to regulate functions described bellow. Here, we summarize, in angiogenesis and cancer, the various functional roles played by p120-RasGAP domains and their effector partners in downstream signaling.

Depletion of the novel protein PHACTR-1 from human endothelial cells abolishes tube formation and induces cell death receptor apoptosis

Using suppression subtractive hybridisation (SSH), we identified a hitherto unreported gene PHACTR-1 (Phosphatase Actin Regulating Protein-1) in Human Umbilical Vascular Endothelial Cells (HUVECs). PHACTR-1 is an actin and protein phosphatase 1 (PP1) binding protein which is reported to be highly expressed in brain and which controls PP1 activity and F-actin remodelling. We have also reported that its expression is dependent of Vascular Endothelial Growth Factor (VEGF-A(165)). To study its function in endothelial cells, we used a siRNA strategy against PHACTR-1. PHACTR-1 siRNA-treated HUVECs showed a major impairment of tube formation and stabilisation. PHACTR-1 depletion triggered apoptosis through death receptors DR4, DR5 and FAS, which was reversed using death receptor siRNAs or with death receptor-dependent caspase-8 siRNA. Our findings suggest that PHACTR-1 is likely to be a key regulator of endothelial cell function properties. Because of its central role in the control of tube formation and endothelial cell survival, PHACTR-1 may represent a new target for the development of anti-angiogenic therapy.

Neuropilin-1 regulates a new VEGF-induced gene, Phactr-1, which controls tubulogenesis and modulates lamellipodial dynamics in human endothelial cells

Recently, we identified a new Vascular Endothelial Growth Factor (VEGF)-A(165)-induced gene Phactr-1, (Phosphatase Actin Regulator-1). We reported that Phactr-1 gene silencing inhibited tube formation in human umbilical endothelial cells (HUVECs) indicating a key role for Phactr-1 in tubulogenesis in vitro. In this study, we investigated the role of Phactr-1 in several cellular processes related to angiogenesis. We found that neuropilin-1 (NRP-1) and VEGF-R1 depletion inhibited Phactr-1 mRNA expression while NRP-2 and VEGF-R2 depletion had no effect. We described a new interaction site of VEGF-A(165) to VEGF-R1 in peptides encoded by exons 7 and 8 of VEGF-A(165). The specific inhibition of VEGF-A(165) binding on NRP-1 and VEGF-R1 by ERTCRC and CDKPRR peptides decreased the Phactr-1 mRNA levels in HUVECs indicating that VEGF-A(165)-dependent regulation of Phactr-1 expression required both NRP-1 and VEGF-R1 receptors. In addition, upon VEGFA(165)-stimulation Phactr-1 promotes formation and maintenance of cellular tubes through NRP-1 and VEGFR1. Phactr-1 was previously identified as protein phosphatase 1 (PP1) α-interacting protein that possesses actin-binding domains. We showed that Phactr-1 depletion decreased PP1 activity, disrupted the fine-tuning of actin polymerization and impaired lamellipodial dynamics. Taken together our results strongly suggest that Phactr-1 is a key component in the angiogenic process.

Synthesis of novel diarylamino-1,3,5-triazine derivatives as FAK inhibitors with anti-angiogenic activity.

We report herein the synthesis of novel diarylamino-1,3,5-triazine derivatives as FAK (focal adhesion kinase) inhibitors and the evaluation of their anti-angiogenic activity on HUVEC cells. Generally, the effects of these compounds on endothelial cells could be correlated with their kinase inhibitory activity. The most efficient compounds displayed inhibition of viability against HUVEC cells in the micromolar range, as observed with TAE-226, which was designed by Novartis Pharma AG. X-ray crystallographic analysis of the co-crystal structure for compound 34 revealed that the mode of interaction with the FAK kinase domain is highly similar to that observed in the complex of TAE-226.

A RasGAP SH3 peptide aptamer inhibits RasGAP-Aurora interaction and induces caspase-independent tumor cell death.

The Ras GTPase-activating protein RasGAP catalyzes the conversion of active GTP-bound Ras into inactive GDP-bound Ras. However, RasGAP also acts as a positive effector of Ras and exerts an anti-apoptotic activity that is independent of its GAP function and that involves its SH3 (Src homology) domain. We used a combinatorial peptide aptamer approach to select a collection of RasGAP SH3 specific ligands. We mapped the peptide aptamer binding sites by performing yeast two-hybrid mating assays against a panel of RasGAP SH3 mutants. We examined the biological activity of a peptide aptamer targeting a pocket delineated by residues D295/7, L313 and W317. This aptamer shows a caspase-independent cytotoxic activity on tumor cell lines. It disrupts the interaction between RasGAP and Aurora B kinase. This work identifies the above-mentioned pocket as an interesting therapeutic target to pursue and points its cognate peptide aptamer as a promising guide to discover RasGAP small-molecule drug candidates.

Inhibition of both focal adhesion kinase and fibroblast growth factor receptor 2 pathways induces anti-tumor and anti-angiogenic activities

FAK and FGFR2 signaling pathways play important roles in cancer development, progression and tumor angiogenesis. PHM16 is a novel ATP competitive inhibitor of FAK and FGFR2. To evaluate the therapeutic efficacy of this agent, we examined its anti-angiogenic effect in HUVEC and its anti-tumor effect in different cancer cell lines. We showed PHM16 inhibited endothelial cell viability, adherence and tube formation along with the added ability to induce endothelial cell apoptosis. This compound significantly delayed tumor cell growth. Together, these data showed that inhibition of both FAK and FGFR2 signaling pathways can enhance anti-tumor and anti-angiogenic activities.

Toll-like receptor 3 regulates cord blood-derived endothelial cell function in vitro and in vivo

Circulating endothelial progenitor cells (cEPC) are capable of homing to neovascularisation sites, in which they proliferate and differentiate into endothelial cells. Transplantation of cEPC-derived cells, in particular those isolated from umbilical cord blood (UCB), has emerged as a promising approach in the treatment of cardio-vascular diseases. After in vivo transplantation, these cells may be exposed to local or systemic inflammation or pathogens, of which they are a common target. Because Toll-like receptors (TLR) are critical in detecting pathogens and in initiating inflammatory responses, we hypothesized that TLR may govern UCB cEPC-derived cells function. While these cells expressed almost all TLR, we found that only TLR3 dramatically impaired cell properties. TLR3 activation inhibited cell proliferation, modified cell cycle entry, impaired the in vitro angiogenic properties and induced pro-inflammatory cytokines production. The anti-angiogenic effect of TLR3 activation was confirmed in vivo in a hind-limb ischemic mice model. Moreover, TLR3 activation consistently leads to an upregulation of miR-29b, -146a and -155 and to a deregulation of cytoskeleton and cell cycle regulator. Hence, TLR3 activation is likely to be a key regulator of cEPC-derived cells properties.

RasV12 induces Survivin/AuroraB pathway conferring tumor cell apoptosis resistance

Several cancers are treated by interferons alpha and gamma in association with conventional chemotherapy due to the resistance observed with interferon treatment alone. The frequency of un-sensitive cancer depends on tumor origin and oncogenic genes. Preclinical studies have highlighted interferon resistance in many cancers such as colon carcinoma due to oncogenic Ras. However, the resistance mechanism remains elusive. Apoptosis and proliferation of Ras(wt) and mutated Ras(V12) transformed colon carcinoma cells treated with several recombinant interferon combinations were analyzed by flow cytometer and immunoblot. Apoptotic pathways of resistant Ras(V12) cells were investigated using siRNA strategy to determine key proteins involved in this process. We show that interferons alpha and gamma synergized to induce human Ras(wt) colon carcinoma cell (HT29) apoptosis by caspases and PARP-1 cleavages in contrast to Ras(V12) mutated colon carcinoma cells (SW480, HT29 clone). However, Ras(V12) siRNA restored interferon sensitivity of Ras(V12)-HT29 clone to apoptosis. Survivin siRNA increased interferon apoptosis in Ras(wt) cells demonstrating the key role of this protein in cell survival. Ras(V12) mutation in HT29 clone neutralized the interferon effect on Survivin suppression and maintained high level of phospho-Aurora-B/Histone H3, which protected cells from apoptosis. SiRNA strategy against both Aurora-B and Survivin in Ras(V12) cells synergized to restore interferon -induced apoptosis. Ras(V12) cells are less sensitive than Ras(wt) cells to interferon induced cell apoptosis due to a Survivin/Aurora-B survival alternative pathway. Taken together, these results may provide interest in siRNA-therapeutic strategy and diagnostic relevance for therapy.

Abstract 3293: Leptin induces breast cancer metastasis through a Neuropilin-1 (NRP-1)/OBR complex

Several studies have demonstrated Neuropilin-1 (NRP-1) implication in tumor progression independently of its known co-receptors. On the basis of growing connections between breast cancer and obesity, we postulated that leptin and its receptor OBR could be potential candidates. First, using NRP-1 over-expression in NRP-1 negative T47D or NRP-1 silencing in NRP-1 positive MDA-MB231 xenografts, we demonstrated that leptin induced metastasis in lymph node only in NRP-1 positive cells. Second, we demonstrated by co-immunoprecipitation, Proximity Ligation assay (duolink) and BRET analysis that NRP-1 is a co-receptor of OBR. Upon leptin exposure this complex is internalized to the nucleus. At this level, NRP-1 Chip-Seq analysis generated from MDA-MB-231 led to identify sequences of RNA polymerase II (Pol2) and transcription factor binding sites of genes implicated in breast cancer metastasis. This finding was confirmed by the detection of NRP-1/Pol2 interaction and by transcriptome analysis of RNA extracts from T47D-NRP-1 xenografts that shows modulation of these genes. In conclusion, we have demonstrated that NRP-1 is a co-receptor of OBR and upon leptin exposure may play a role as a transcription factor regulating genes involved in cancer progression. Thus, NRP-1 might be a therapeutic target to prevent cancer metastasis induced by leptin Citation Format: Zakia Belaid-Choucair, Julie Dam, Nicolas Goudin, Odile Filhole, Claude Cochet, Yves lepelletier, Tereza Coman, Genevieve Courtois, Alain Colige, Marie-Paule Defresne, Ralf Jockers, Olivier Hermine. Leptin induces breast cancer metastasis through a Neuropilin-1 (NRP-1)/OBR complex. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3293. doi:10.1158/1538-7445.AM2014-3293