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Figure S1 shows Mesenchymal traits of human MDA-B02 and BC-M1 breast cancer cells. Figure S2 shows Quantitative RT-PCR measurements of miR-30 expression levels. Figure S3 shows Quantification of absolute amounts of miR-30s. Figure S4 shows Stable overexpression of miR-30 family members in human MDA-B02 breast cancer cells. Figure S5 shows miR-30s expression in MDA-B02 and BC-M1 breast cancer cells. Figure S6 shows Western blot analyses. Figure S7 shows that CDH11 is a target for repression by miR-30s. Figure S8 shows Interaction of MDA-MB-231 and Hs-578T silenced for CDH11 with MC3T3-E1 osteoblasts. Figure S9 shows ITGB3 and CTGF are direct targets for repression by miR-30s. Figure S10 shows Putative binding sites between miR-30s and the ITGA5 3'UTR. Figure S11 shows effects of miR-30s on MCF-7 tumor cell colonization of the bone marrow ex vivo.

Legends to Supplementary Figures S1 to S11.

miRNAs are master regulators of gene expression that play key roles in cancer metastasis. During bone metastasis, metastatic tumor cells must rewire their biology and express genes that are normally expressed by bone cells (a process called osteomimicry), which endow tumor cells with full competence for outgrowth in the bone marrow. Here, we establish miR-30 family members miR-30a, miR-30b, miR-30c, miR-30d, and miR-30e as suppressors of breast cancer bone metastasis that regulate multiple pathways, including osteomimicry. Low expression of miR-30 in primary tumors from patients with breast cancer were associated with poor relapse-free survival. In addition, estrogen receptor (ER)-negative/progesterone receptor (PR)-negative breast cancer cells expressed lower miR-30 levels than their ER/PR-positive counterparts. Overexpression of miR-30 in ER/PR-negative breast cancer cells resulted in the reduction of bone metastasis burden in vivo. In vitro, miR-30 did not affect tumor cell proliferation, but did inhibit tumor cell invasion. Furthermore, overexpression of miR-30 restored bone homeostasis by reversing the effects of tumor cell–conditioned medium on osteoclastogenesis and osteoblastogenesis. A number of genes associated with osteoclastogenesis stimulation (IL8, IL11), osteoblastogenesis inhibition (DKK-1), tumor cell osteomimicry (RUNX2, CDH11), and invasiveness (CTGF, ITGA5, ITGB3) were identified as targets for repression by miR-30. Among these genes, silencing CDH11 or ITGA5 in ER-/PR-negative breast cancer cells recapitulated inhibitory effects of miR-30 on skeletal tumor burden in vivo. Overall, our findings provide evidence that miR-30 family members employ multiple mechanisms to impede breast cancer bone metastasis and may represent attractive targets for therapeutic intervention.Significance: These findings suggest miR-30 family members may serve as an effective means to therapeutically attenuate metastasis in triple-negative breast cancer. Cancer Res; 78(18); 5259–73. ©2018 AACR.

Description of (1) retroviral vector constructs, (2) ShRNA design sequences, (3) psiCHECK-2 vector constructs, (4) co-culture of breast tumor cells with osteoblasts, (5) breast cancer cohort of patients, (6) miRNA quantification in primary tumor of breast cancer patients and (7) miRNA profiling of breast cancer cell lines.

In breast cancer bone metastasis, tumor cells stimulate osteoclast-mediated bone resorption, and bone-derived growth factors released from resorbed bone stimulate tumor growth. The αvβ3 integrin is an adhesion receptor expressed by breast cancer cells and osteoclasts. It is implicated in tumor cell invasion and osteoclast-mediated bone resorption. Here, we hypothesized that the therapeutic targeting of tumor αvβ3 integrin would prevent bone metastasis formation. We first showed that, compared with mock-transfected cells, the i.v. inoculation of αvβ3-overexpressing MDA-MB-231 breast cancer cells in animals increased bone metastasis incidence and promoted both skeletal tumor burden and bone destruction. The direct inoculation of αvβ3-overexpressing transfectants into the tibial bone marrow cavity did not however enhance skeletal tumor burden and bone destruction, suggesting that αvβ3 controls earlier events during bone metastasis formation. We next examined whether a nonpeptide antagonist of αvβ3 (PSK1404) exhibits meaningful antitumor effects in experimental breast and ovarian cancer bone metastasis. A continuous PSK1404 treatment, which inhibited osteoclast-mediated bone resorption in an animal model of bone loss, substantially reduced bone destruction and decreased skeletal tumor burden. Importantly, a short-term PSK1404 treatment that did not inhibit osteoclast activity also decreased skeletal tumor burden and bone destruction. This dosing regimen caused a profound and specific inhibition of bone marrow colonization by green fluorescent protein, αvβ3-expressing tumor cells in vivo and blocked tumor cell invasion in vitro. Overall, our data show that tumor αvβ3 integrin stands as a therapeutic target for the prevention of skeletal metastases. [Cancer Res 2007;67(12):5821–30]

Supplementary Table 2 from Bone Morphogenetic Protein 7 in the Development and Treatment of Bone Metastases from Breast Cancer

Supplementary Table 1 from Bone Morphogenetic Protein 7 in the Development and Treatment of Bone Metastases from Breast Cancer

Supplementary Methods and Materials from Bone Morphogenetic Protein 7 in the Development and Treatment of Bone Metastases from Breast Cancer

Supplementary Figures 1-3 from Tumor αvβ3 Integrin Is a Therapeutic Target for Breast Cancer Bone Metastases

Bone morphogenetic protein 7 (BMP7) counteracts the physiological epithelial-to-mesenchymal transition (EMT), a process that is indicative of epithelial plasticity. Because EMT is involved in cancer, we investigated whether BMP7 plays a role in breast cancer growth and metastasis. In this study, we show that decreased BMP7 expression in primary breast cancer is significantly associated with the formation of clinically overt bone metastases in patients with ≥10 years of follow-up. In line with these clinical observations, BMP7 expression is inversely related to tumorigenicity and invasive behavior of human breast cancer cell lines. Moreover, BMP7 decreased the expression of vimentin, a mesenchymal marker associated with invasiveness and poor prognosis, in human MDA-MB-231 (MDA-231)-B/Luc+ breast cancer cells under basal and transforming growth factor-β (TGF-β)–stimulated conditions. In addition, exogenous addition of BMP7 to TGF-β–stimulated MDA-231 cells inhibited Smad-mediated TGF-β signaling. Furthermore, in a well-established bone metastasis model using whole-body bioluminescent reporter imaging, stable overexpression of BMP7 in MDA-231 cells inhibited de novo formation and progression of osteolytic bone metastases and, hence, their metastatic capability. In line with these observations, daily i.v. administration of BMP7 (100 μg/kg/d) significantly inhibited orthotopic and intrabone growth of MDA-231-B/Luc+ cells in nude mice. Our data suggest that decreased BMP7 expression during carcinogenesis in the human breast contributes to the acquisition of a bone metastatic phenotype. Because exogenous BMP7 can still counteract the breast cancer growth at the primary site and in bone, BMP7 may represent a novel therapeutic molecule for repression of local and bone metastatic growth of breast cancer. [Cancer Res 2007;67(18):8742–51]

Supplementary Figure Legends 1-3 from Tumor αvβ3 Integrin Is a Therapeutic Target for Breast Cancer Bone Metastases

Supplementary Figure 1 from Bone Morphogenetic Protein 7 in the Development and Treatment of Bone Metastases from Breast Cancer

International audience; Objective: To explore the regulatory role of soluble CD146 (sCD146) and its interaction with galectin-1 (Gal1) in placenta-mediated complications of pregnancy.Design: Prospective pilot and experimental studies.Setting: University-affiliated hospital and academic research laboratory.Patient(s): One hundred fifteen women divided into three groups: 30 healthy, nonpregnant women, 50 women with normal pregnancies, and 35 with placenta-mediated pregnancy complications.Intervention(s): Wound-healing experiments were conducted to study trophoblast migration.Main outcome measure(s): Quantification of sCD146 and Gal1 by enzyme-linked immunosorbent assay. Analysis of trophoblast migration by wound closure.Result(s): Concomitant detection of sCD146 and Gal1 showed lower sCD146 and higher Gal1 concentrations in women with normal pregnancies compared with nonpregnant women. In addition, follow-up of these women revealed a decrease in sCD146 associated with an increase in Gal1 throughout pregnancy. In contrast, in women with preeclampsia, we found significantly higher sCD146 concentrations compared with women with normal pregnancies and no modification of Gal1. We emphasize the opposing effects of sCD146 and Gal, since, unlike Gal1, sCD146 inhibits trophoblast migration. Moreover, the migratory effect of Gal1 was abrogated with the use of an anti-CD146 blocking antibody or the use of small interfering RNA to silence VEGFR2 expression. This suggests that trophoblast migration is mediated though the interaction of Gal1 with CD146, further activating the VEGFR2 signaling pathway. Significantly, sCD146 blocked the migratory effects of Gal1 on trophoblasts and inhibited its secretion, suggesting that sCD146 acts as a ligand trap.Conclusion(s): Soluble CD146 could be proposed as a biomarker in preeclampsia and a potential therapeutic target. Clinical trial registration number: NCT 01736826.Trial registration: ClinicalTrials.gov NCT01736826.

International audience; CD146 involvement was recently described in skin fibrosis of systemic sclerosis through its regulation of the Wnt pathway. Because the interaction between Wnt and ROS signaling plays a major role in fibrosis, we hypothesized that in systemic sclerosis, CD146 may regulate Wnt/ROS crosstalk. Using a transcriptomic and western blot analysis performed on CD146 wild-type or knockout mouse embryonic fibroblasts, we showed a procanonical Wnt hallmark in the absence of CD146 that is reversed when CD146 expression is restored. We found an elevated ROS content in knockout cells and an increase in DNA oxidative damage in the skin sections of knockout mice compared with those of wild-type mice. We also showed that ROS increased CD146 and its noncanonical Wnt ligand, WNT5A, only in wild-type cells. In humans, fibroblasts from patients with systemic sclerosis presented higher ROS content and expressed CD146, whereas control fibroblasts did not. Moreover, CD146 and its ligand were upregulated by ROS in both human fibroblasts. The increase in bleomycin-induced WNT5A expression was abrogated when CD146 was silenced. We showed an interplay between Wnt and ROS signaling in systemic sclerosis, regulated by CD146, which promotes the noncanonical Wnt pathway and prevents ROS signaling, opening the way for innovative therapeutic strategies.

Background: Triple Negative Breast Cancers (TNBC) are the most aggressive breast cancers and lead to poor prognoses. This is due to a high resistance to therapies, mainly because of the presence of Cancer Stem Cells (CSCs). Plasticity, a feature of CSCs, is acquired through the Epithelial to Mesenchymal Transition (EMT), a process that has been recently shown to be regulated by a key molecule, CD146. Of interest, CD146 is over-expressed in TNBC. Methods: The MDA-MB-231 TNBC cell line was used as a model to study the role of CD146 and its secreted soluble form (sCD146) in the development and dissemination of TNBC using in vitro and in vivo studies. Results: High expression of CD146 in a majority of MDA-MB-231 cells leads to an increased secretion of sCD146 that up-regulates the expression of EMT and CSC markers on the cells. These effects can be blocked with a specific anti-sCD146 antibody, M2J-1 mAb. M2J-1 mAb was able to reduce tumour development and dissemination in a model of cells xenografted in nude mice and an experimental model of metastasis, respectively, in part through its effects on CSC. Conclusion: We propose that M2J-1 mAb could be used as an additional therapeutic approach to fight TNBC.

International audience; Objective Systemic sclerosis (SSc) is an autoimmune disorder characterized by excessive fibrosis, immune dysfunction, and vascular damage, in which the expression of many growth factors is deregulated. CD146 was recently described as a major actor in SSc. Since CD146 also exists as a circulating soluble form (sCD146) that acts as a growth factor in numerous angiogenic- and inflammation-related pathologies, we sought to identify the mechanisms underlying the generation of sCD146 and to characterize the regulation and functions of the different variants identified in SSc. Methods We performed in vitro experiments, including RNA-Seq and antibody arrays, and in vivo experiments using animal models of bleomycin-induced SSc and hind limb ischemia. Results Multiple forms of sCD146, generated by both shedding and alternative splicing of the primary transcript, were discovered. The shed form of sCD146 was generated from the cleavage of both long and short membrane isoforms of CD146 through ADAM-10 and TACE metalloproteinases, respectively. In addition, 2 novel sCD146 splice variants, I5-13-sCD146 and I10-sCD146, were identified. Of interest, I5-13-sCD146 was significantly increased in the sera of SSc patients (P < 0.001; n = 117), in particular in patients with pulmonary fibrosis (P < 0.01; n = 112), whereas I10-sCD146 was decreased (P < 0.05; n = 117). Further experiments revealed that shed sCD146 and I10-sCD146 displayed proangiogenic activity through the focal adhesion kinase and protein kinase C epsilon signaling pathways, respectively, whereas I5-13-sCD146 displayed profibrotic effects through the Wnt-1/beta-catenin/WISP-1 pathway. Conclusion Variants of sCD146, and in particular the novel I5-13-sCD146 splice variant, could constitute novel biomarkers and/or molecular targets for the diagnosis and treatment of SSc and other angiogenesis- or fibrosis-related disorders.

International audience; CD146 is an endothelial junctional adhesion molecule, which expression is increased in human glomerular diseases. However, the pathological significance of this overexpression remains unknown. Induction of glomerulonephritis in mice, by using nephrotoxic serum, showed that CD146 expression was highly induced within damaged glomeruli and was associated with renal inflammation and fibrosis. Interestingly, 2 weeks after glomerulonephritis induction, CD146 knockout mice showed preserved renal function as proteinuria and blood urea nitrogen levels were significantly lower compared with wild-type littermates. Furthermore, renal structure was considerably conserved, since crescents formation, tubular dilation, monocyte and lymphocyte infiltration, and interstitial renal fibrosis were highly reduced. Colocalization with markers for different types of glomerular cells showed that CD146 expression was mainly increased within the injured endothelium of the glomerular tuft. Consequently, we generated a new transgenic strain in which CD146 was specifically deleted in the vascular endothelium. Similarly to CD146 knockout, these mice showed preservation of renal structure and function after the induction of glomerulonephritis compared with wild-type animals. These data show that endothelial CD146 plays a major role in glomerulonephritis and may represent a novel therapeutic target to reduce glomerular damage and the progression of renal disease.

International audience; Initially discovered in human melanoma, CD146/MCAM is expressed on many tumors and is correlated with cancer progression and metastasis. However, targeting CD146 remains challenging since it is also expressed on other cell types, as vessel cells, where it displays important physiological functions. We previously demonstrated that CD146 is shed as a soluble form (sCD146) that vectorizes the effects of membrane CD146 on tumor angiogenesis, growth and survival. We thus generated a novel monoclonal antibody, the M2J-1 mAb, which specifically targets sCD146, but not membrane CD146, and counteracts these effects. In our study, we analyzed the effects of sCD146 on the dissemination and the associated procoagulant phenotype in two highly invasive human CD146-positive cancer cell lines (ovarian and melanoma). Results show that sCD146 induced epithelial to mesenchymal transition, favored the generation of cancer stem cells and increased the membrane expression of tissue factor. Treatment of cancer cells with sCD146 in two experimental models (subcutaneous xenografting and intracardiac injection of cancer cells in nude mice) led to increased tumor dissemination and procoagulant activity. The M2J-1 mAb drastically reduced metastasis but also procoagulant activity, in particular by decreasing the number of circulating tumor microparticles, and blocked the relevant signaling pathways as demonstrated by RNA expression profiling experiments. Thus, our findings demonstrate that sCD146 mediates important pro-metastatic and procoagulant effects in two CD146-positive tumors. Targeting sCD146 with the newly generated M2J-1 mAb could constitute an innovative strategy for preventing dissemination and thromboembolism in many CD146-positive tumors.

International audience; E dema is the main symptom of nephrotic syndrome associated to minimal change disease (MCD). Besides the increase in glomerular permeability, possibly induced by a circulating permeability factor, 1 an increase in systemic vascular permeability could participate in the constitution of edema. This study was conducted to evaluate the permeability of endothelial cells (EC) exposed to serum from nephrotic patients with MCD, to low-or high-molecularweight molecules, by trans-or paracellular transport.

International audience; Aims: The progression of atherosclerosis is based on the continued recruitment of leukocytes in the vessel wall. The previously described role of CD146 in leukocyte infiltration suggests an involvement for this adhesion molecule in the inflammatory response. In this study, we investigated the role of CD146 in leukocyte recruitment by using an experimental model of atherogenesis.Methods and results: The role of CD146 was explored in atherosclerosis by crossing CD146-/- mice with ApoE-/- mice. CD146 -/-/ApoE -/- and ApoE -/- mice were fed a Western diet for 24 weeks and were monitored for aortic wall thickness using high frequency ultrasound. The arterial wall was significantly thicker in CD146-deficient mice. After 24 weeks of Western diet, a significant increase of atheroma in both total aortic lesion and aortic sinus of CD146-null mice was observed. In addition, atherosclerotic lesions were more inflammatory since plaques from CD146-deficient mice contained more neutrophils and macrophages. This was due to up-regulation of RANTES secretion by macrophages in CD146-deficient atherosclerotic arteries. This prompted us to further address the function of CD146 in leukocyte recruitment during acute inflammation by using a second experimental model of peritonitis induced by thioglycollate. Neutrophil recruitment was significantly increased in CD146-deficient mice 12 h after peritonitis induction and associated with higher RANTES levels in the peritoneal cavity. In CD146-null macrophages, we also showed that increased RANTES production was dependent on constitutive inhibition of the p38-MAPK signaling pathway. Finally, Maraviroc, a RANTES receptor antagonist, was able to reduce atherosclerotic lesions and neutrophilia in CD146-deficient mice to the same level as that found in ApoE -/- mice.Conclusions: Our data indicate that CD146 deficiency is associated with the upregulation of RANTES production and increased inflammation of atheroma, which could influence the atherosclerotic plaque fate. Thus, these data identify CD146 agonists as potential new therapeutic candidates for atherosclerosis treatment.

International audience; CD146 (cluster of differentiation 146) is an adhesion molecule that is expressed by different cells constituting vessels, particularly endothelial cells. The last 30 years of research in this field have shown that CD146 plays a key role in the control of several vessel functions. Three forms of CD146 have been described, including 2 transmembrane isoforms and a soluble protein that is detectable in the plasma. These CD146 forms mediate pleiotropic functions through homophilic and heterophilic interactions with proteins present on surrounding partners. Several studies used neutralizing antibodies, siRNA, or genetically modified mice to demonstrate the involvement of CD146 in the regulation of angiogenesis, vascular permeability, and leukocyte transmigration. In this review, we will focus on the current knowledge of the roles of CD146 in vascular homeostasis and diseases associated with endothelial dysfunction.

Cell-based therapies constitute a real hope for the treatment of ischaemic diseases. One of the sources of endothelial progenitors for autologous cell therapy is Endothelial Colony Forming Cells (ECFC) that can be isolated from peripheral blood. However, their use is limited by their low number in the bloodstream and the loss of their stem cell phenotype associated with the acquisition of a senescent phenotype in culture. We hypothesized that adding soluble CD146, a novel endothelial growth factor with angiogenic properties, during the isolation and growth procedures could improve their number and therapeutic potential. Soluble CD146 increased the number of isolated peripheral blood ECFC colonies and lowered their onset time. It prevented cellular senescence, induced a partial mesenchymal phenotype and maintained a stem cell phenotype by stimulating the expression of embryonic transcription factors. These different effects were mediated through the induction of mature miR-21. When injected in an animal model of hindlimb ischaemia, sCD146-primed ECFC isolated from 40 ml of blood from patients with peripheral arterial disease were able to generate new blood vessels and restore blood flow. Treatment with sCD146 could thus constitute a promising strategy to improve the use of autologous cells for the treatment of ischaemic diseases.

miRNAs are master regulators of gene expression that play key roles in cancer metastasis. During bone metastasis, metastatic tumor cells must rewire their biology and express genes that are normally expressed by bone cells (a process called osteomimicry), which endow tumor cells with full competence for outgrowth in the bone marrow. Here, we establish miR-30 family members miR-30a, miR-30b, miR-30c, miR-30d, and miR-30e as suppressors of breast cancer bone metastasis that regulate multiple pathways, including osteomimicry. Low expression of miR-30 in primary tumors from patients with breast cancer were associated with poor relapse-free survival. In addition, estrogen receptor (ER)-negative/progesterone receptor (PR)-negative breast cancer cells expressed lower miR-30 levels than their ER/PR-positive counterparts. Overexpression of miR-30 in ER/PR-negative breast cancer cells resulted in the reduction of bone metastasis burden in vivo. In vitro, miR-30 did not affect tumor cell proliferation, but did inhibit tumor cell invasion. Furthermore, overexpression of miR-30 restored bone homeostasis by reversing the effects of tumor cell–conditioned medium on osteoclastogenesis and osteoblastogenesis. A number of genes associated with osteoclastogenesis stimulation (IL8, IL11), osteoblastogenesis inhibition (DKK-1), tumor cell osteomimicry (RUNX2, CDH11), and invasiveness (CTGF, ITGA5, ITGB3) were identified as targets for repression by miR-30. Among these genes, silencing CDH11 or ITGA5 in ER-/PR-negative breast cancer cells recapitulated inhibitory effects of miR-30 on skeletal tumor burden in vivo. Overall, our findings provide evidence that miR-30 family members employ multiple mechanisms to impede breast cancer bone metastasis and may represent attractive targets for therapeutic intervention. Significance: These findings suggest miR-30 family members may serve as an effective means to therapeutically attenuate metastasis in triple-negative breast cancer. Cancer Res; 78(18); 5259–73. ©2018 AACR.

International audience; Senescent cells may exert detrimental effect on microenvironment through the secretion of soluble factors and the release of extracellular vesicles, such as microparticles, key actors in ageing and cardiovascular diseases. We previously reported that sirtuin-1 (SIRT1) deficiency drives accelerated senescence and dysfunction of endothelial colony-forming cells (ECFC) in PT neonates. Because preterm birth (PT) increases the risk for cardiovascular diseases during neonatal period as well as at adulthood, we hypothesized that SIRT1 deficiency could control the biogenesis of microparticles as part of a senescence–associated secretory phenotype (SASP) of PT-ECFC and investigated the related molecular mechanisms. Compared to control ECFC, PT-ECFC displayed a SASP associated with increased release of endothelial microparticles (EMP), mediating a paracrine induction of senescence in naïve endothelial cells. SIRT1 level inversely correlated with EMP release and drives PT-ECFC vesiculation. Global transcriptomic analysis revealed changes in stress response pathways, specifically the MAPK pathway. We delineate a new epigenetic mechanism by which SIRT1 deficiency regulates MKK6/p38 MAPK /Hsp27 pathway to promote EMP biogenesis in senescent ECFC. These findings deepen our understanding of the role of ECFC senescence in the disruption of endothelial homeostasis and provide potential new targets towards the control of cardiovascular risk in individuals born preterm. Preterm (PT) birth is associated with an increased risk of vascular-related diseases during the neonatal period, such as bronchopulmonary dysplasia and retinopathy, as well as early in adulthood, including hypertension and emphysema 1–4. Adverse early programming may explain such association and influence cardiovascular risk over the life course by acting on the vascular bed. Vascular alterations are major determinants and include an increase in arterial stiffness, a reduction in microvascular density because of incomplete vasculogenesis, and an impairment of endothelial function 5, 6. Endothelial Colony Forming Cells (ECFC) are a relevant subset of endothelial progenitor cells that mainly reside in vessel wall, circulate at low frequency in the blood, and support vascular repair and de novo vessel formation 7–9. A growing body of evidence supports their role as potential biomarkers and therapeutic targets in PT birth 5, 10, 11. Among the factors that can alter cord blood ECFC functions in premature neonates, we recently demonstrated the

CD146 is an adhesion molecule expressed by both melanoma and endothelial cells and thus is well positioned to control melanoma extravasation. Nevertheless, during melanoma metastasis, the involvement of CD146 expressed within tumor microenvironment has never been analyzed. To investigate whether host CD146 mediates the extravasation of melanoma cells across the endothelium, we generated CD146 KO mice. We demonstrated that host CD146 did not affect melanoma growth or tumor angiogenesis but promoted hematogenous melanoma metastasis to the lung. Accordingly, the survival of CD146-deficient mice was markedly prolonged during melanoma metastasis. Interestingly, vascular endothelial growth factor-induced vascular permeability was significantly decreased in CD146 KO mice. We also provided evidence that VEGF-induced transendothelial migration of melanoma cells was significantly reduced across CD146 KO lung microvascular endothelial cells (LMEC). CD146 deficiency decreased the expression of VEGFR-2/Ve-cadherin and altered focal adhesion kinase (FAK) activation in response to VEGF. In addition, inhibition of FAK phosphorylation reduced transmigration of B16 melanoma cells across WT LMEC at the same level that across CD146 KO LMEC. Altogether, we propose a novel mechanism involving the VEGF/CD146/FAK/Ve-cadherin network in melanoma extravasation across the vessel barrier that identifies CD146-targeted therapy as a potential strategy for the treatment of melanoma metastasis.

International audience; We highlight for the first time that CD146/sCD146 is involved in fibrotic process during SSc. sCD146 could thus constitute a new biomarker to assess disease activity, and potentially a new target for therapeutic applications.

CD146 is an adhesion molecule present on many tumors (melanoma, kidney, pancreas, breast, ...). In addition, it has been shown to be expressed on vascular endothelial and smooth muscle cells. Generating an antibody able to specifically recognize CD146 in cancer cells (designated as tumor CD146), but not in normal cells, would thus be of major interest for targeting tumor CD146 without affecting the vascular system. We thus generated antibodies against the extracellular domain of the molecule produced in cancer cells and selected an antibody that specifically recognizes tumor CD146. This antibody (TsCD146 mAb) was able to detect CD146-positive tumors in human biopsies and in vivo, by PET imaging, in a murine xenograft model. In addition, TsCD146 mAb antibody was able to specifically detect CD146-positive cancer microparticles in the plasma of patients. TsCD146 mAb displayed also therapeutic effects since it was able to reduce the growth of human CD146-positive cancer cells xenografted in nude mice. This effect was due to a decrease in the proliferation and an increase in the apoptosis of CD146-positive cancer cells after TsCD146-mediated internalization of the cell surface CD146. Thus, TsCD146 mAb could be of major interest for diagnostic and therapeutic strategies against CD146-positive tumors in a context of personalized medicine.

The clinical efficacy of anti-angiogenic monotherapies in metastatic breast cancer is less than originally anticipated, and it is not clear what the response of bone metastasis to anti-angiogenic therapies is. Here, we examined the impact of neutralizing tumor-derived vascular endothelial growth factor (VEGF) in animal models of subcutaneous tumor growth and bone metastasis formation. Silencing of VEGF expression (Sh-VEGF) in osteotropic human MDA-MB-231/B02 breast cancer cells led to a substantial growth inhibition of subcutaneous Sh-VEGF B02 tumor xenografts, as a result of reduced angiogenesis, when compared to that observed with animals bearing mock-transfected (Sc-VEGF) B02 tumors. However, there was scant evidence that either the silencing of tumor-derived VEGF or the use of a VEGF-neutralizing antibody (bevacizumab) affected B02 breast cancer bone metastasis progression in animals. We also examined the effect of vatalanib (a VEGF receptor tyrosine kinase inhibitor) in this mouse model of bone metastasis. However, vatalanib failed to inhibit bone metastasis caused by B02 breast cancer cells. In sharp contrast, vatalanib in combination with bevacizumab reduced not only bone destruction but also skeletal tumor growth in animals bearing breast cancer bone metastases, when compared with either agent alone. Thus, our study highlights the importance of targeting both the tumor compartment and the host tissue (i.e., skeleton) to efficiently block the development of bone metastasis. We believe this is a crucially important observation as the clinical benefit of anti-angiogenic monotherapies in metastatic breast cancer is relatively modest.

International audience; CD146 (MUC-18, MCAM) expression on cancer cells correlates with cancer progression and a bad prognosis in several tumors, including melanoma and pancreatic tumors. Deciphering the mechanism mediating the CD146 role in cancer is essential for generating new therapeutic strategies. We found that CD146 expression in cancer cells is associated with a secretion of soluble CD146 (sCD146) that constitutes an active player in tumor development. Indeed, sCD146 induces the overexpression of its binding protein, angiomotin, on both endothelial and cancer cells and promotes both paracrine effects on angiogenesis and autocrine effects on cancer cells proliferation and survival. These last effects are mediated in part through the induction and phosphorylation of c-myc in cancer cells. In mice models xenografted with human CD146-positive melanoma or pancreatic cancer cells, administration of a novel monoclonal antibody specifically targeting sCD146, but not its membrane form, successfully suppresses tumor vascularization and growth. Our findings demonstrate that sCD146 secreted by CD146-positive tumors mediates important pro-angiogenic and pro-tumoral effects. Targeting sCD146 with a novel neutralizing antibody could thus constitute an innovative therapeutic strategy for the treatment of CD146-positive tumors.

International audience; Aims: Endothelial colony-forming cells (ECFC) constitute an endothelial progenitor fraction with a promising interest for the treatment of ischaemic cardiovascular diseases. As soluble CD146 (sCD146) is a new factor promoting angiogenesis, we examined whether sCD146 priming could improve the therapeutic potential of ECFC and defined the involved mechanism.Methods and results: We investigated the effects of sCD146 priming on regenerative properties of ECFC in vivo. In a mouse model of hindlimb ischaemia, the homing of radiolabelled cells to ischaemic tissue was assessed by SPECT-CT imaging. Soluble CD146 priming did not modify the number of engrafted ECFC but improved their survival capacity, leading to an enhanced revascularization. The mechanism of action of sCD146 on ECFC was studied in vitro. We showed that sCD146 acts in ECFC through a signalosome, located in lipid rafts, containing angiomotin, the short isoform of CD146 (shCD146), VEGFR1, VEGFR2, and presenilin-1. Soluble CD146 induced a sequential proteolytic cleavage of shCD146, with an extracellular shedding followed by an intramembrane cleavage mediated by matrix metalloprotease (MMP)/ADAM and presenilin-1, respectively. The generated intracellular part of shCD146 was directed towards the nucleus where it associated with the transcription factor CSL and modulated the transcription of genes involved in cell survival (FADD, Bcl-xl) and angiogenesis (eNOS). This effect was dependent on both VEGFR1 and VEGFR2, which were rapidly phosphorylated by sCD146.Conclusions: These findings establish that activation of the proteolytic processing of shCD146, in particular by sCD146, constitutes a promising pathway to improve endothelial progenitors' regenerative properties for the treatment of cardiovascular diseases.

Rationale: The progression of atherosclerosis is based on the continued recruitment of leukocytes in the vessel wall. The previously described role of CD146 in leukocyte infiltration in vitro suggests a role for this endothelial junction molecule in the pathogenesis of atherosclerosis. However, its involvement in atherosclerotic plaques formation has never been investigated. Objective: We evaluated the role of CD146 in atherogenesis. Methods and Results: CD146 -/-/ApoE -/- and ApoE -/- mice were fed a Western diet for 24 weeks and were monitored for aortic wall thickness using high frequency ultrasound. The arterial wall thickness was significantly higher in CD146 deficient mice. We evidenced a significant increase of atheroma in both total aortic lesion and aortic sinus of CD146 deficient mice. In addition, atherosclerotic lesions were more inflammatory since CD146 deficient plaques contained more neutrophils and more macrophages. During atherosclerosis, circulating neutrophils were significantly increased in the absence of CD146. Consistent with the higher recruitment of inflammatory cells to the atheroma, we demonstrated that RANTES was up-regulated in CD146 deficient atherosclerotic arteries. In addition, CD146 deficient mice presented significant higher levels of circulating RANTES during atherosclerosis. Finally, we showed that macrophages were the source of RANTES and its production by CD146 null macrophages was also significantly increased through a mechanism dependent of p38-MAPK signaling pathway. Conclusions: Our data indicate that CD146 deficiency is associated with the upregulation of RANTES and increased inflammation of atheroma, which could influence the atherosclerotic plaque fate. Thus, these data identify CD146 as a potential new target for atherosclerosis treatment.

Introduction La maladie de Still ou forme systemique de l’arthrite juvenile idiopathique (FS-AJI) est une maladie dependante de l’IL-1s. Cependant, cette cytokine pro-inflammatoire qui suit des voies de secretion inhabituelles et mal connues, est souvent non mesurable dans le serum des patients. Les microparticules (MPs) leucocytaires pourraient constituer un mecanisme de secretion de l’IL-1s, mais leurs fonctions biologiques ainsi que leur pertinence clinique dans des maladies comme la FS-AJI n’ont jamais ete demontrees. Materiels et methodes Nous avons etudie la presence de l’IL-1s, des elements de l’inflammasome NLRP3 et du facteur tissulaire (FT) au sein de MPs issues de la stimulation de THP-1 ou de cellules mononucleees peripheriques sanguines (PBMCs) differenciees en macrophages par Elisa, western-blot ou cytometrie de flux. Les fonctions biologiques des MPs etaient etudiees in vitro par l’expression de molecules d’adhesion leuco-endotheliales, la generation de facteur Xa et in vivo, dans un modele animal d’inflammation neutrophilique. Enfin, nous avons etudie l’existence de MPs circulantes chez des patients atteints de FS-AJI. Resultats La stimulation des macrophages par du LPS et de l’ATP entraine la production de MPs de grandes tailles (> 500 nm) qui contiennent le precurseur et la forme mature de l’IL-1s, l’inflammasome NLRP3, la caspase-1 et le FT biologiquement actif. Ces MPs expriment le recepteur P2X7 et liberent de l’IL-1s soluble apres activation par l’ATP in vitro. Les MPs contenant l’IL-1s induisent in vitro, l’expression de molecules d’adhesion a la surface endotheliale et in vivo, une inflammation neutrophilique peritoneale en partie dependante de l’IL-1. Compares aux temoins sains, les patients atteints de FS-AJI presentent un profil microparticulaire altere avec une augmentation significative des MPs leucocytaires CD45+ et CD11b+ et des grandes MPs contenant l’IL-1s. Conclusion Les macrophages actives liberent des microparticules pro-inflammatoires et pro-thrombotiques, contenant l’IL-1s et l’inflammasome NLRP3 qui peuvent etre detectees chez des patients atteints de forme systemique d’arthrite juvenile idiopathique.

MicroRNAs (miRNAs), which are master regulators of gene expression, have emerged as key players in cancer metastasis. Here, we established miR-30 family members (miR-30a, miR-30b, miR-30c, miR-30d and miR-30e) as breast cancer bone metastasis suppressor genes. Low expression levels of miR-30s in primary tumors of breast cancer patients were associated with poor relapse-free survival. Additionally, miR-30s levels were significantly lower in estrogen receptor (ER)-negative/progesterone receptor (PR)-negative tumors than in their hormone-responsive counterparts. Overexpression of miR-30s in ER/PR-negative breast cancer cells resulted in the reduction of bone metastasis burden in vivo. MiR-30s overexpression also reduced tumor outgrowth, when tumor cells were implanted subcutaneously in animals. In vitro, miR-30s did not affect tumor cell proliferation, but inhibited tumor cell invasion. Furthermore, miR-30s restored bone homeostasis by reversing the in vitro effects of the tumor cell conditioned medium on stimulation of osteoclastogenesis and inhibition of osteoblastogenesis. We identified a number of genes associated with osteoclastogenesis stimulation (IL-8, IL-11), osteoblastogenesis inhibition (DKK-1), tumor cell osteomimicry (RUNX2, CDH11) and invasiveness (CTGF, ITGA5, ITGB3) that were direct and/or indirect targets for repression by miR-30s. Among these genes, integrin ITGA5 was a previously unknown miR-30 target. By silencing ITGA5 in ER-/PR-negative breast cancer cells, colonization of the bone marrow by tumor cells was drastically reduced in vivo. Overall, our data indicate that miR-30s employ multiple mechanisms to impede breast cancer bone metastasis. These findings may pave the way to a new field of therapeutic interventions in breast cancer patients with bone metastasis. Citation Format: Martine Croset, Francesco Pantano, Casina Kan, Edith Bonnelye, Francoise Descotes, Catherine Alix-Panabières, Charles Lecellier, Richard Bachelier, Nathalie Allioli, Saw See Hong, Kai Bartkowiak, Klaus Pantel, Philippe Clézardin. MicroRNA-30 family members inhibit breast cancer invasion, osteomimicry, and bone destruction by directly targeting multiple bone metastasis-associated genes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 486.

In breast cancer bone metastasis, tumor cells stimulate osteoclast-mediated bone resorption, and bone-derived growth factors released from resorbed bone stimulate tumor growth. The αvβ3 integrin is an adhesion receptor expressed by breast cancer cells and osteoclasts. It is implicated in tumor cell invasion and osteoclast-mediated bone resorption. Here, we hypothesized that the therapeutic targeting of tumor αvβ3 integrin would prevent bone metastasis formation. We first showed that, compared with mock-transfected cells, the i.v. inoculation of αvβ3-overexpressing MDA-MB-231 breast cancer cells in animals increased bone metastasis incidence and promoted both skeletal tumor burden and bone destruction. The direct inoculation of αvβ3-overexpressing transfectants into the tibial bone marrow cavity did not however enhance skeletal tumor burden and bone destruction, suggesting that αvβ3 controls earlier events during bone metastasis formation. We next examined whether a nonpeptide antagonist of αvβ3 (PSK1404) exhibits meaningful antitumor effects in experimental breast and ovarian cancer bone metastasis. A continuous PSK1404 treatment, which inhibited osteoclast-mediated bone resorption in an animal model of bone loss, substantially reduced bone destruction and decreased skeletal tumor burden. Importantly, a short-term PSK1404 treatment that did not inhibit osteoclast activity also decreased skeletal tumor burden and bone destruction. This dosing regimen caused a profound and specific inhibition of bone marrow colonization by green fluorescent protein, αvβ3-expressing tumor cells in vivo and blocked tumor cell invasion in vitro. Overall, our data show that tumor αvβ3 integrin stands as a therapeutic target for the prevention of skeletal metastases. [Cancer Res 2007;67(12):5821–30]

Atherosclerosis is a chronic inflammatory disease of the large vessels and its progression is based in part on the continued recruitment of macrophages in the inflammatory wall. The well described role of CD146 in leukocyte infiltration in vitro suggests a role for this endothelial junction molecule in the early stages of the formation of atherosclerotic plaques. However, the involvement of CD146 in atherogenesis has not been elucidated. To investigate the role of CD146 deficiency in atherosclerosis, CD146 knocked-out (KO) mice were bred with ApoE KO mice. Mice were fed a high-fat Western diet for 24 weeks and were monitored for aortic wall thickness using high frequency ultrasound. After 24 weeks of diet, the arterial wall thickness was significantly higher in CD146 deficient mice (0.19±0.01mm vs 0.27±0.01mm; p=0.012) and there was a significant 47 % increase (p=0.015) in total aortic lesion area, evaluated by histological oil red O coloration. However, mice weights and total cholesterol plasma levels were similar between the two groups of mice. We detected a 2-fold increase in macrophage recruitment to the atherosclerotic plaque of the double KO mice compared with ApoE KO mice, suggesting that the atherosclerotic lesions in these mice were more inflammatory. In addition, CD146 deficient plaques contained significantly less smooth muscle cells (p=0.036) and could be more vulnerable. We observed a maladaptive immune response in double KO mice since the circulating neutrophils and monocytes were significantly increased (p=0.008 and p=0.015 respectively) in the absence of CD146 whereas circulating T lymphocytes were significantly decreased (p=0.026). In a similar way, splenocytes isolated from double KO mice contained significantly lower levels of T cells (p=0.002), suggesting a default of adaptive immunity in the absence of CD146 during atherosclerosis. Our results demonstrated the involvement of CD146 in the progression of atherosclerosis in mice. Our data also suggest that CD146 is involved in the regulation of adaptive immune response and its absence lead to an inflammatory phenotype, which could influence atherosclerosis plaque fate. Thus, CD146 should be considered as a potential target for protection against atherosclerosis.

CD146 is an adhesion molecule expressed by both melanoma and endothelial cells and thus is well positioned to control melanoma extravasation. Nevertheless, during melanoma metastasis, the involvement of CD146 expressed within tumor microenvironment has never been analyzed. To investigate whether host CD146 mediates the extravasation of melanoma cells across the endothelium, we generated CD146 KO mice. We demonstrated that host CD146 did not affect melanoma growth or tumor angiogenesis but promoted hematogenous melanoma metastasis to the lung. Accordingly, the survival of CD146-deficient mice was markedly prolonged during melanoma metastasis. Interestingly, vascular endothelial growth factor-induced vascular permeability was significantly decreased in CD146 KO mice. We also provided evidence that VEGF-induced transendothelial migration of melanoma cells was significantly reduced across CD146 KO lung microvascular endothelial cells (LMEC). CD146 deficiency decreased the expression of VEGFR-2/Ve-cadherin and altered focal adhesion kinase (FAK) activation in response to VEGF. In addition, inhibition of FAK phosphorylation reduced transmigration of B16 melanoma cells across WT LMEC at the same level that across CD146 KO LMEC. Altogether, we propose a novel mechanism involving the VEGF/CD146/FAK/Ve-cadherin network in melanoma extravasation across the vessel barrier that identifies CD146-targeted therapy as a potential strategy for the treatment of melanoma metastasis.

Epidemiological and experimental studies indicate that early vascular dysfunction occurs in low-birth-weight subjects, especially preterm (PT) infants. We recently reported impaired angiogenic activity of endothelial colony-forming cells (ECFCs) in this condition. We hypothesized that ECFC dysfunction in PT might result from premature senescence and investigated the underlying mechanisms. Compared with ECFCs from term neonates (n = 18), ECFCs isolated from PT (n = 29) display an accelerated senescence sustained by growth arrest and increased senescence-associated β-galactosidase activity. Increased p16(INK4a) expression, in the absence of telomere shortening, indicates that premature PT-ECFC aging results from stress-induced senescence. SIRT1 level, a nicotinamide adenine dinucleotide-dependent deacetylase with anti-aging activities, is dramatically decreased in PT-ECFCs and correlated with gestational age. SIRT1 deficiency is subsequent to epigenetic silencing of its promoter. Transient SIRT1 overexpression or chemical induction by resveratrol treatment reverses senescence phenotype, and rescues in vitro PT-ECFC angiogenic defect in a SIRT1-dependent manner. SIRT1 overexpression also restores PT-ECFC capacity for neovessel formation in vivo. We thus demonstrate that decreased expression of SIRT1 drives accelerated senescence of PT-ECFCs, and acts as a critical determinant of the PT-ECFC angiogenic defect. These findings lay new grounds for understanding the increased cardiovascular risk in individuals born prematurely and open perspectives for therapeutic strategy.

BRCA1 mutations are involved in breast and ovarian cancer predisposition in humans. The biological functions of the murine BRCA1 gene have been extensively studied but little is known about murine BRCA1 proteins. To better characterize these proteins, we have cloned the full-length murine BRCA1 cDNA and a splice variant deleted of exon 11, BRCA1-Δ11, by RT-PCR method. Three polyclonal antibodies raised against various parts of murine BRCA1 were used in our study: D16, M20 and 5MO, which were generated in our laboratory. This allowed us to analyze the expression and subcellular localization of both isoforms in murine and human cell lines by immunoblotting, immunoprecipitation, cell fractionation and immunofluorescence. Endogenous BRCA1 was detected in murine cell lines but not splice variant BRCA1-Δ11, whereas both ectopically expressed murine isoforms were detected in transfected human Bosc 23 cells. Subcellular fractionation and immunofluorescence results showed that the BRCA1 protein was mainly located in the nucleus, whereas BRCA1-Δ11 was preferentially cytoplasmic. The conservation of exon 11 splicing and the differential subcellular localization of BRCA1 and BRCA1-Δ11 in human and mouse suggest that these proteins could play distinct roles and that they could differentially act in the pathological mechanisms leading to the development of breast and ovarian cancer. The characterization of the murine BRCA1 proteins and antibodies will be useful to further study BRCA1 functions in murine models. Int. J. Cancer 88:519–524, 2000. © 2000 Wiley-Liss, Inc.

The clinical efficacy of anti-angiogenic monotherapies in metastatic breast cancer is less than originally anticipated, and it is not clear what the response of bone metastasis to anti-angiogenic therapies is. Here, we examined the impact of neutralizing tumor-derived vascular endothelial growth factor (VEGF) in animal models of subcutaneous tumor growth and bone metastasis formation. Silencing of VEGF expression (Sh-VEGF) in osteotropic human MDA-MB-231/B02 breast cancer cells led to a substantial growth inhibition of subcutaneous Sh-VEGF B02 tumor xenografts, as a result of reduced angiogenesis, when compared to that observed with animals bearing mock-transfected (Sc-VEGF) B02 tumors. However, there was scant evidence that either the silencing of tumor-derived VEGF or the use of a VEGF-neutralizing antibody (bevacizumab) affected B02 breast cancer bone metastasis progression in animals. We also examined the effect of vatalanib (a VEGF receptor tyrosine kinase inhibitor) in this mouse model of bone metastasis. However, vatalanib failed to inhibit bone metastasis caused by B02 breast cancer cells. In sharp contrast, vatalanib in combination with bevacizumab reduced not only bone destruction but also skeletal tumor growth in animals bearing breast cancer bone metastases, when compared with either agent alone. Thus, our study highlights the importance of targeting both the tumor compartment and the host tissue (i.e., skeleton) to efficiently block the development of bone metastasis. We believe this is a crucially important observation as the clinical benefit of anti-angiogenic monotherapies in metastatic breast cancer is relatively modest.

Bone morphogenetic protein 7 (BMP7) counteracts the physiological epithelial-to-mesenchymal transition (EMT), a process that is indicative of epithelial plasticity. Because EMT is involved in cancer, we investigated whether BMP7 plays a role in breast cancer growth and metastasis. In this study, we show that decreased BMP7 expression in primary breast cancer is significantly associated with the formation of clinically overt bone metastases in patients with ≥10 years of follow-up. In line with these clinical observations, BMP7 expression is inversely related to tumorigenicity and invasive behavior of human breast cancer cell lines. Moreover, BMP7 decreased the expression of vimentin, a mesenchymal marker associated with invasiveness and poor prognosis, in human MDA-MB-231 (MDA-231)-B/Luc+ breast cancer cells under basal and transforming growth factor-β (TGF-β)–stimulated conditions. In addition, exogenous addition of BMP7 to TGF-β–stimulated MDA-231 cells inhibited Smad-mediated TGF-β signaling. Furthermore, in a well-established bone metastasis model using whole-body bioluminescent reporter imaging, stable overexpression of BMP7 in MDA-231 cells inhibited de novo formation and progression of osteolytic bone metastases and, hence, their metastatic capability. In line with these observations, daily i.v. administration of BMP7 (100 μg/kg/d) significantly inhibited orthotopic and intrabone growth of MDA-231-B/Luc+ cells in nude mice. Our data suggest that decreased BMP7 expression during carcinogenesis in the human breast contributes to the acquisition of a bone metastatic phenotype. Because exogenous BMP7 can still counteract the breast cancer growth at the primary site and in bone, BMP7 may represent a novel therapeutic molecule for repression of local and bone metastatic growth of breast cancer. [Cancer Res 2007;67(18):8742–51]