Search

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 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.

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.

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]

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.

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]

Breast cancer patients with advanced disease exhibit bone metastases, leading to the formation of osteolytic lesions for which the only currently available treatments are palliative. Here, we describe how we refined a mouse model of human breast cancer metastasis into bone, characterized its transcriptome and demonstrated its clinical relevance. Cells were selected from bone metastases caused by MDA-MB-231 cells after several in vivo passages, and engineered to express luciferase. Whole body bioluminescence live imaging indicated that the selected isogenic B02 clone was unique in its ability to form rapidly growing osteolytic bone metastases. B02 cells were detected as early as 10 days after tail vein injection, as opposed to 1 month after cardiac injection in other haematogenous models. Whole transcriptomic analysis identified 114 upregulated and 247 downregulated genes in B02 cells compared to the parental cells, several of which represent novel targets. In addition, there was a 50% overlap between the B02 signature and a recently described signature obtained from human breast cancer bone metastases. Consistent with the plasticity of an aggressive metastatic variant, 10% of the regulated genes are involved in proliferation, migration, invasion and angiogenesis. Strikingly, B02 cells also express osteoblast-specific genes, thus mimicking a process referred to as osteomimicry in the clinic. The B02 cells “human bone metastatic signature”, the expression of bone-specific genes, as well as the live imaging of this convenient model highlight its clinical relevance and usefulness during drug development.

Germline mutations of breast cancer-associated gene 1 (BRCA1) predispose women to breast and ovarian cancer. It was recently shown that bilateral oophorectomy decreases breast cancer incidence in BRCA1 mutation carriers. To model human BRCA1 carriers, our laboratory has previously created mice with a conditional knockout of the full-length BRCA1 gene in the mammary epithelium combined with a heterozygous knockout of the p53 tumor suppressor gene. These mice developed ER-negative mammary tumors and were employed to determine the effects of oophorectomy on tumor formation. Individual knockout mice (BRCA1(Co/Co) MMTV-Crep53+/-), following two complete pregnancies, were either oophorectomized or sham treated. Mice were subsequently examined for the development of palpable mammary tumors until they were 12 months of age. Until 135 days post-oophorectomy (255 days of age), the tumor incidence was similar in both oophorectomized and intact mice, approximately 30%. After this time, the increase in tumor incidence was much lower in the oophorectomized mice, while tumor incidence increased in non-oophorectomized mice. The effects of oophorectomy on mammary development in both control and knockout mice were also examined. Oophorectomized mice with a conditional knockout of full-length BRCA1 in conjunction with a loss of one p53 allele exhibited glandular regression with a reduction in the number of mammary epithelial cells following oophorectomy. This study employed a model that may be relevant for testing agents useful against breast cancer in BRCA1 carriers and a subset of sporadic cancers. The data also show that oophorectomy, if performed significantly prior to the time that tumors arise, appears to be quite effective.

Breast-cancer-associated gene 1 (BRCA1) is highly expressed in thymus and spleen. In this paper, we have studied lymphocyte development and tumorigenesis in mice carrying mutations in Brca1 and p53. We show that the deletion of Brca1 exon 11 (Brca1-delta11), which disrupts the full-length isoform, but not the short isoform of Brca1, does not interfere with lymphocyte development. This is true irrespective of p53 status, that is, whether it is wild type, heterozygous or homozygous for a null mutation. These data suggest that the expression of Brca1 short isoform alone is enough to maintain normal development of lymphocytes. However, it cannot suppress tumorigenesis as about 30% of Brca1(delta11/delta11)p53(+/-) mice develop thymic lymphoma between 3 and 7 months of age. We demonstrate that p53 plays an essential role in Brca1-associated lymphoma, as all the tumors from Brca1(delta11/delta11)p53(+/-) mice exhibit LOH of p53 and Brca1(delta11/delta11)p53(-/-) mice exhibited accelerated tumorigenesis. We further demonstrate that the Brca1-delta11 deficiency does not affect thymocyte proliferation; however, it increases genetic instability and triggers gamma-irradiation-induced apoptosis. The loss of p53 attenuates apoptosis and allows accumulation of further mutations in Brca1-delta11 thymocytes, eventually leading to thymic lymphoma formation.

Germ-line alterations in BRCA1 are associated with an increased susceptibility to breast and ovarian cancer. BRCA1 is a 220-kDa protein that contains a tandem of two BRCA1 C-Terminal (BRCT) domains. Among missense and nonsense BRCA1 mutations responsible for family breast cancer, some are located into the BRCT tandem of BRCA1 coding sequence. In an attempt to understand how BRCT is critical for BRCA1 function, we search for partners of this BRCT tandem of BRCA1. Using a glutathione-S-transferase (GST) pull-down assay with murine cells, we isolated only one major BRCA1-interacting protein, further identified as Acetyl Coenzyme A (CoA) Carboxylase alpha (ACCA). We showed that this interaction is conserved through murine and human species. We also delineated the minimum interacting region as being the whole tandem of BRCT domains. We demonstrated that BRCA1 interacts in vitro and in vivo with ACCA. This interaction is completely abolished by five distinct germline BRCA1 deleterious mutations affecting the BRCT tandem of BRCA1. Interestingly, ACCA originally known as a rate-limiting enzyme for fatty acids biosynthesis, has been recently shown to be over-expressed in breast cancers and considered as a potential target for anti-neoplastic therapy. Furthermore, our observation is making a bridge between the genetic factors involved in susceptibility to breast and ovarian cancers, and environmental factors such as nutrition considered as key elements in the etiology of those cancers.

We have investigated the effects of the expression of wild-type and mutant Brca1 alleles on the murine mammary gland morphogenesis and carcinogenesis. Primary cultures of mammary cells from BALB/cByJIco mice were infected with recombinant Babe Puro retroviruses expressing lacZ, full-length Brca1, splice variant Brca1-Delta11, or mutant Brca1-W1777Stop alleles. Infected cells were reinjected into the mammary fat pad of a syngeneic virgin mouse whose endogenous epithelium had previously been removed. Four months after reinjection, nulliparous and postlactating mice were checked for the reconstitution of the mammary gland. Stable expression of beta-galactosidase was observed in the ducts formed by epithelial mammary cells infected with Babe Puro/ lacZ retrovirus. Epithelial mammary cells transduced with full-length Brca1 developed normally, whereas those transduced with Brca1-Delta11 or Brca1-W1777Stop formed atypical duct hyperplasia associated with reduced branching. These results suggest that ectopically expressed splice variant Brca1-Delta11 and mutant Brca1-W1777Stop have dominant negative effects.

Angiogenesis is essential for sustained tumour growth and various factors such as VEGF and VEGF receptors have been found to play an essential role in this process. However, little is known about the role of VEGF on bone metastasis formation. To address this question, VEGF-A expression was silenced in the BO2 cell line, an osteotropic variant of the human breast cancer cell line MDA-MB-231. An 80–90% decrease of VEGF levels was found in several BO2 clones, as determined by RT/qPCR and ELISA. These clones were then injected in nude mice either subcutaneously for tumourigenesis or intravenously for bone metastasis experiments. Although VEGF silencing was efficient at inhibiting the subcutaneous growth of tumour xenografts, it did not significantly reduce bone metastasis formation. To target the VEGF/VEGFR pathway in the tumour cells and the bone microenvironment, we then decided to treat mice injected with luciferase-expressing BO2 cells with two different drugs: Avastin, a monoclonal antibody directed against human VEGF-A (5 mg/kg given i.p. every 3 days) and PTK787, a tyrosine kinase inhibitor of VEGFR-1 and VEGFR2 (100 mg/kg given daily per os). Treatments were administered according to two different settings: preventive (from day 0 to day 28) or curative (from day 14 to day 28). The extent of skeletal tumour burden and bone destruction in animals was monitored 13, 21 and 28 days after tumour cell injection using bioluminescence imaging and X-ray radiography, respectively. Avastin or PTK787, when used as single agents, did not inhibit bone metastasis formation. In contrast, a combined treatment using Avastin + PTK787 inhibited the development of osteolytic lesions in both the preventive and curative settings. However, a combined treatment did not affect the skeletal tumour burden. Overall, these results provide evidence that VEGF-A and its receptors play an important role during bone metastasis formation.