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Supplementary Table S1. Primer pairs used for quantitative real-time PCR analysis. Supplementary Table S2. Summary of the gene expression datasets used for meta-analysis. Supplementary Table S3. Expression levels of LOX-like mRNAs in Hct116 cells transduced for LOX overexpression (LOX+) or silencing (LOX-), compared to mock-transduced Hct116 cells (Ctrl).

Lysyl oxidase (LOX) is a secreted copper-dependent amine oxidase whose primary function is to drive collagen crosslinking and extracellular matrix stiffness. LOX in colorectal cancer synergizes with hypoxia-inducible factor-1 (HIF-1) to promote tumor progression. Here we investigated whether LOX/HIF1 endows colorectal cancer cells with full competence for aggressive colonization in bone. We show that a high LOX expression in primary tumors from patients with colorectal cancer was associated with poor clinical outcome, irrespective of HIF-1. In addition, LOX was expressed by tumor cells in the bone marrow from colorectal cancer patients with bone metastases. In vivo experimental studies show that LOX overexpression in colorectal cancer cells or systemic delivery of the conditioned medium from LOX-overexpressing colorectal cancer cells promoted tumor cell dissemination in the bone marrow and enhanced osteolytic lesion formation, irrespective of HIF-1. Conversely, silencing or pharmacologic inhibition of LOX activity blocked dissemination of colorectal cancer cells in the bone marrow and tumor-driven osteolytic lesion formation. In vitro, tumor-secreted LOX supported the attachment and survival of colorectal cancer cells to and in the bone matrix, and inhibited osteoblast differentiation. LOX overexpression in colorectal cancer cells also induced a robust production of IL6. In turn, both LOX and IL6 were acting in concert to promote RANKL-dependent osteoclast differentiation, thereby creating an imbalance between bone resorption and bone formation. Collectively, our findings show that LOX supports colorectal cancer cell dissemination in the bone marrow and they reveal a novel mechanism through which LOX-driven IL6 production by colorectal cancer cells impairs bone homeostasis. Cancer Res; 77(2); 268–78. ©2016 AACR.

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.

Supplementary figure legends from S1 to S7

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.

Figure S1: LOX expression and clinical outcomes. Figure S2:The LOX inhibitor βAPN inhibits osteolytic lesion formation in vivo. Figure S3: Measurement of LOX enzymatic activity. Figure S4: Correlation analysis of the expression intensity of LOX and IL-6. Figure S5: Effects of LOX expression on EMT in Hct116 colorectal cancer cells. Figure S6: Effect of LOX on attachment of colorectal cancer cells to fibronectin. Figure S7: LOX enhances survival of Hct116 colorectal cancer cells.

Bone metastasis remains a major cause of mortality and morbidity in breast cancer. Therefore, there is an urgent need to better select high-risk patients in order to adapt patient’s treatment and prevent bone recurrence. Here, we found that integrin alpha5 (ITGA5) was highly expressed in bone metastases, compared to lung, liver, or brain metastases. High ITGA5 expression in primary tumors correlated with the presence of disseminated tumor cells in bone marrow aspirates from early stage breast cancer patients (n = 268; p = 0.039). ITGA5 was also predictive of poor bone metastasis-free survival in two separate clinical data sets (n = 855, HR = 1.36, p = 0.018 and n = 427, HR = 1.62, p = 0.024). This prognostic value remained significant in multivariate analysis (p = 0.028). Experimentally, ITGA5 silencing impaired tumor cell adhesion to fibronectin, migration, and survival. ITGA5 silencing also reduced tumor cell colonization of the bone marrow and formation of osteolytic lesions in vivo. Conversely, ITGA5 overexpression promoted bone metastasis. Pharmacological inhibition of ITGA5 with humanized monoclonal antibody M200 (volociximab) recapitulated inhibitory effects of ITGA5 silencing on tumor cell functions in vitro and tumor cell colonization of the bone marrow in vivo. M200 also markedly reduced tumor outgrowth in experimental models of bone metastasis or tumorigenesis, and blunted cancer-associated bone destruction. ITGA5 was not only expressed by tumor cells but also osteoclasts. In this respect, M200 decreased human osteoclast-mediated bone resorption in vitro. Overall, this study identifies ITGA5 as a mediator of breast-to-bone metastasis and raises the possibility that volociximab/M200 could be repurposed for the treatment of ITGA5-positive breast cancer patients with bone metastases.

The oncogenic transcription factor ZNF217 orchestrates several molecular signaling networks to reprogram integrated circuits governing hallmark capabilities within cancer cells. High levels of ZNF217 expression provide advantages to a specific subset of cancer cells to reprogram tumor progression, drug resistance and cancer cell plasticity. ZNF217 expression level, thus, provides a powerful biomarker of poor prognosis and a predictive biomarker for anticancer therapies. Cancer epigenetic mechanisms are well known to support the acquisition of hallmark characteristics during oncogenesis. However, the complex interactions between ZNF217 and epigenetic processes have been poorly appreciated. Deregulated DNA methylation status at ZNF217 locus or an intricate cross-talk between ZNF217 and noncoding RNA networks could explain aberrant ZNF217 expression levels in a cancer cell context. On the other hand, the ZNF217 protein controls gene expression signatures and molecular signaling for tumor progression by tuning DNA methylation status at key promoters by interfering with noncoding RNAs or by refining the epitranscriptome. Altogether, this review focuses on the recent advances in the understanding of ZNF217 collaboration with epigenetics processes to orchestrate oncogenesis. We also discuss the exciting burgeoning translational medicine and candidate therapeutic strategies emerging from those recent findings connecting ZNF217 to epigenetic deregulation in cancer.

Introduction L’osteogenese imparfaite (OI) est une maladie osseuse rare a l’origine de fractures et de deformations osseuses. Dans 85 % des cas, elle est due a une mutation autosomique dominante de COL1A1 ou COL1A2. Pour un meme gene mute et parfois pour le meme variant, il existe une grande variabilite de severite phenotypique, sans aucun biomarqueur pour le praticien a ce jour. En tant que regulateurs epigenetiques de l’expression des genes, les microARNs (miARNs) circulants ont ete decrits dans d’autres maladies osseuses comme etant de potentiels marqueurs pronostiques. L’objectif de mirOI est d’identifier des miARNs circulant potentiellement associes a la severite de l’OI. Patients et methodes Nous avons realise cette etude en 3 etapes. Nous avons tout d’abord identifie, par RNA-sequencing d’un petit echantillon de patients OI versus controles, 19 microARNs d’interet sur- ou sous-exprimes significativement entre les deux groupes. Nous avons ensuite confirme cela sur un echantillon plus important par RT-qPCR. Nous avons enfin cherche une relation entre niveau de variation des miARNs confirmes avec les caracteristiques cliniques de l’OI. Resultats Au cours de la premiere phase de miROI, nous mettons en evidence 79 miARNs differentiellement exprimes entre les deux groupes de faibles effectifs (10 OI versus 10 temoins) de maniere significative avec une valeur de p corrigee inferieure a 0.05 et 27 miARNs avec un p corrige inferieur a 0.0005. Nous en avons donc selectionne 19 qui nous semblaient les plus pertinents (resultats presentes en 2020). Dans la deuxieme phase nous sommes parvenus a confirmer dans un plus grand effectif (49 OI versus 49 controles sains apparies sur âge, sexe et IMC) la surexpression significative de 8 miARNs dans le groupe OI. Enfin, la comparaison de la variation du niveau d’expression de ces miARNs aux donnees cliniques met en evidence une difference significative de deux d’entre eux dans les OI presentant une dentinogenese imparfaite. Nous retrouvons egalement une correlation negative faible entre plusieurs miARNs et certains items type « sante mentale » du questionnaire SF36. En dehors de ces parametres nous n’avons pas retrouve d’association significative entre la variation du niveau d’expression et des criteres de severite de la maladie plus durs. Apres revue de la litterature, nous avons retrouve 6 parmi les 8 miARNs deja connus pour avoir une action directe sur l’homeostasie osseuse. Par ailleurs, l’utilisation d’un modele de prediction d’interaction miARNs - genes retrouve une probabilite d’interaction a 100 % entre 3 des 8 miARNs confirmes et COL1A1 et/ou COL1A2. Conclusion Au total, MiROI est la premiere etude a etablir la signature miARN de l’OI en mettant en evidence une modification d’expression significative de miARNs, potentiellement impliques dans la regulation de genes jouant un role essentiel, direct ou indirect, dans la physiopathologie de l’OI. Le role des miARNs mis en evidence ici necessitera la realisation d’une etude sur modeles cellulaires et animaux afin de confirmer et etudier leur implication dans la perturbation de l’homeostasie osseuse de l’OI. Cette derniere pourrait deboucher sur l’identification d’une cible concrete pour de futures therapies ciblant les miARNs impliques.

Introduction L’osteogenese imparfaite (OI) est une maladie osseuse rare a l’origine de fractures et de deformations osseuses. Dans 85 % des cas, elle est due a une mutation autosomique dominante de COL1A1 ou COL1A2. Pour un meme gene mute (COL1A1 ou COL1A2) et pour le meme variant (dans une meme famille par exemple), il existe une grande variabilite de severite phenotypique, sans aucun biomarqueur pour le praticien. En tant que regulateurs epigenetiques de l’expression des genes, les microARNs (miARNs) circulants ont ete decrits dans d’autres maladies osseuses comme etant de potentiels marqueurs pronostiques. L’objectif est d’identifier des miARNs circulant potentiellement associes a la severite de l’OI. Materiels et methodes 10 patients presentant une OI ont ete recrutes en consultation ou hospitalisation dans notre service. Dix controles, apparies sur l’âge et le sexe, ont ete inclus a partir des cohortes OFELY, MODAM, STRAMBO. Le profil des miARNs circulants (miRome) a ete etabli par Next Generation Sequencing (NGS) apres l’extraction de l’ARN total suivi du sequencage des miARNs sur la plateforme Illumina (QIAGEN Genomic Services). Le niveau d’expression des miARNs a ete normalise par TMM (Trimmed Mean of M), exprime en TPM (Transcripts Per Million) et compare entre chaque groupe par le ratio de TPM exprime en donnees relatives (Log2Fold Change). Du fait des comparaisons multiples, nous avons applique la correction du False Discovery Rate (FDR) selon la methode de Benjamini-Hochberg. Resultats L’âge moyen des patients OI est de 41 ans avec un sex-ratio equilibre. Tous les patients presentent une OI de type I. Nous mettons en evidence 79 miARNs differentiellement exprimes entre les deux groupes de maniere significative avec une valeur de p corrigee par FDR inferieure a 0,05 et 27 miARNs avec un p corrige inferieur a 0,0005. Parmi eux, 22 sont sur-exprimes et 5 sont sous-exprimes. Nous avons realise une revue de litterature montrant que 22 parmi ces 27 miARNs sont connus pour etre impliques dans l’homeostasie osseuse. Nous mettons egalement en evidence des clusters de miARNs, c’est-a-dire des miARNs physiquement adjacents sur l’ADN, entierement sur-exprimes, comme les clusters miR-183/96/182 et miR-25/93/106b. L’utilisation de la base de donnees MiRWALK revele que 27/79 miARNs ciblent potentiellement COL1A1 ou COL1A2 avec une probabilite de 100 %. Conclusion Nous avons etabli ici la signature miARN de l’OI de type 1 en mettant en evidence une modification d’expression significative de miARNs potentiellement impliques dans la regulation de genes ayant un role essentiel dans la physiopathologie de l’OI. Ces miARNs seront soumis a une phase de validation par RT-qPCR et de comparaison a la severite clinique sur un plus large effectif de serums afin de valider leur interet en tant que biomarqueur pronostique de la severite de la maladie.

Fibrous dysplasia (FD) is a rare bone disease caused by activating mutations of GNAS encoding the Gsα protein, enhancing cyclic adenosine monophosphate (cAMP) production by overstimulation of adenylyl cyclase and impairing osteoblastic differentiation. The clinical presentation ranges from asymptomatic to polyostotic forms with severe disability, explained by the mosaic distribution of the GNAS mutation. Physicians have to deal with the gap of knowledge in FD pathogenesis, the absence of prognostic markers and the lack of specific treatment. The identification of specific biomarkers for FD is an important step to improve the clinical and therapeutic approaches. An epigenetic regulation driven by microRNAs (miRNAs), known as promising biomarkers in bone disease, could be involved in FD. We have sought circulating miRNAs that are differentially expressed in FD patients compared to controls and would reflect dysregulations of osteogenesis-related genes and bone disorder. The global miRNA profiling was performed using Next Generation Sequencing in patient serum collected from a discovery cohort of 20 patients (10 polyostotic and 10 monostotic) and 10 controls. From these, we selected 19 miRNAs for a miRNA validation phase from serum of 82 patients and 82 controls, using real-time qPCR. Discovery screening identified 111 miRNAs differentially expressed in patient serum, after adjusting for the false discovery rate (FDR). Among the 82 patients, 55% were polyostotic, and 73% were women with a mean age of 42 years. Six miRNAs (miR-25-3p, miR-93-5p, miR-182-5p, miR-324-5p, miR-363-3p, and miR-451a) were significantly overexpressed in serum, with FDR

Bone metastases contribute substantially to morbidity and mortality in patients with advanced-stage cancers. The metastatic bone disease is driven by tumor cells, which alter the functions of bone-resorbing (osteoclasts) and bone-forming (osteoblasts) cells, promoting skeletal destruction. Pharmacological agents bisphosphonates and denosumab are the standards of care for the treatment of metastatic bone disease. They block bone destruction, thereby delaying the occurrence of bone metastasis and reducing skeletal morbidity. In this review, we describe the progress and future directions of existing bone-targeted therapies and report emerging therapies that have arisen from advances in our understanding of the biology of bone metastases.

ContextMicroRNA (miRNA) regulate post-transcriptionally the expression of osteogenesis and angiogenesis associated genes and emerge as potential non-invasive biomarkers in vascular and bone diseases. Severe abdominal aortic calcification (AAC) is associated with higher risk of cardiovascular event and of fragility fracture.ObjectiveTo identify miRNA linked to the aggravation of AAC and to incident osteoporotic fracture.DesignPostmenopausal women (>50 years) with available serum at inclusion and data for each outcome (Kauppila score and incident fracture) were selected from the OFELY prospective cohort. We conducted a case-control study in 434 age-matched women, 50% with incident osteoporotic fracture over 20 years of follow-up and a second study in 183 women to explore AAC over 17 years.MethodsSerum expression of three miRNA involved in vascular calcification and bone turnover regulation (miRs-26a-5p,-34a-5p, and -223-5p) was quantified at baseline by TaqMan Advanced miRNA technology and expressed by relative quantification. Outcomes were the association of miRNA levels with (1) incident osteoporotic fractures during 20 years, (2) AAC aggravation during 17 years.ResultsMiRNA level was not associated with incident fractures (miR-26a-5p: 1.06 vs 0.99, p = 0.07; miR-34a-5p: 1.15 vs 1.26, p = 0.35; miR-223a-5p: 1.01 vs 1.05, p = 0.32). 93 women had an increase in Kauppila score over 17 years while 90 did not. None of the miRNAs was associated with an aggravation in AAC (miR-26a-5p: 1.09 vs 1.10, p = 0.95; miR-34a-5p: 0.78 vs 0.73, p = 0.90; miR-223-5p: 0.97 vs 0.78, p = 0.11).ConclusionsCirculating miR-26a-5p, -34a-5p and -223-5p are not significantly associated with incident fracture and AAC aggravation.

International audience; Bone is the most common metastatic site for breast cancer. Estrogen-related-receptor alpha (ERRα) has been implicated in cancer cell invasiveness. Here, we established that ERRα promotes spontaneous metastatic dissemination of breast cancer cells from primary mammary tumors to the skeleton. We carried out cohort studies, pharmacological inhibition, gain-of-function analyses in vivo and cellular and molecular studies in vitro to identify new biomarkers in breast cancer metastases. Meta-analysis of human primary breast tumors revealed that high ERRα expression levels were associated with bone but not lung metastases. ERRα expression was also detected in circulating tumor cells from metastatic breast cancer patients. ERRα overexpression in murine 4T1 breast cancer cells promoted spontaneous bone micro-metastases formation when tumor cells were inoculated orthotopically, whereas lung metastases occurred irrespective of ERRα expression level. In vivo, Rank was identified as a target for ERRα. That was confirmed in vitro in Rankl stimulated tumor cell invasion, in mTOR/pS6K phosphorylation, by transactivation assay, ChIP and bioinformatics analyses. Moreover, pharmacological inhibition of ERRα reduced primary tumor growth, bone micro-metastases formation and Rank expression in vitro and in vivo. Transcriptomic studies and meta-analysis confirmed a positive association between metastases and ERRα/RANK in breast cancer patients and also revealed a positive correlation between ERRα and BRCA1 mut carriers. Taken together, our results reveal a novel ERRα/RANK axis by which ERRα in primary breast cancer promotes early dissemination of cancer cells to bone. These findings suggest that ERRα may be a useful therapeutic target to prevent bone metastases.

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.

Background MicroRNAs (miRs) have emerged as pivotal epigenetic key actors of gene regulation and several miRs have been shown to be at the crossroads of angiogenesis and of bone turnover, taking part in the calcification process by acting on osteoblasts and osteoclasts.1 Calcification of the aortic media is highly regulated and involves numerous factors, including calcium deposal and other bone remodelling factors.2 Objectives The objective of this study was to find a signature of miRs linked both to osteoporotic fracture risk and abdominal aortic calcification (AAC). The first outcome was the link between miRs levels at baseline and incident osteoporotic fractures (IOF) during 20 years; the second outcome was the link between miRs levels at baseline and the increase in AAC during 17 years. Methods Post-menopausal women older than 50 years from the OFELY cohort (Os des FEmmes de LYon) were selected if they had available serums at inclusion, and available data for each outcome. 3 miRs selected after literature review because of their impact on vascular calcification and bone turnover (miRs 26a-5p, 34a-5p, and 223–5 p) were measured at baseline. Bioassays of miRs was conducted with miRCURY Biofluids (Exiqon) extraction kit, TaqMan Life Technologies protocol, and QuantStudio 7 flex (Applied Biosystems) for RNA quantification. Results are expressed by relative quantification of Cycle threshold (Ct). Results A sample of 434 age-matched women (63 [57–72] years old), 50% with incident osteoporotic fracture during the 20 years of follow-up, was included. 183 women had available data to explore AAC; 93 had an increase in Kauppila score in 17 years (58 [55–61] years old), 90 did not (55 [53–58] years old). No significant link was underlined between miRs and IOF (miR-26: 1.06 [0.85–1.27] vs 0.99 [0.85–1.17], p=0.07; miR-34: 1.15 [0.53–1.87] vs 1.26 [0.60–2.07], p=0.35; miR-223: 1.01 [0.68–1.43] vs 1 0.05 [0.72–1.56], p=0.32). No miR was significantly linked to an increase in AAC (miR-26: 1.09 [0.94–1.28] vs 1.10 [0.89–1.30], p=0.95; miR-34: 0.78 [0.46–1.21] vs 0.73 [0.38–1.50], p=0.90; miR-223: 0.97 [0.69–1.22] vs 0.78 [0.56–1.22], p=0.11). Conclusions No association was observed between the 3 tested miRs and IOF or increase in AAC. Larger studies are necessary to select interesting epigenetic pathways reproductible on wider population. References [1] Krzeszinskia JY, Wei W, Huynh H, Jin Z, Wang X, Chang T-C, et al. miR-34a Blocks Osteoporosis and Bone Metastasis by Inhibiting Osteoclastogenesis and Tgif2. Nature2014Aug 28;512(7515):431. [2] Szulc P. Abdominal aortic calcification: A reappraisal of epidemiological and pathophysiological data. Bone2016Mar;84:25–37. Disclosure of Interest None declared

Postmenopausal osteoporosis is characterized by the occurrence of fragility fracture with an increase in morbidity and mortality. Recently, microRNAs (miRNAs) have raised interest as regulators of translational repression, mediating a number of key processes, including bone tissue in both physiological and diseased states. The aim of this study was to examine the serum levels of 32 preselected miRNAs with reported function in bone and their association with osteoporotic fracture. We performed cross-sectional and longitudinal analyses from the OFELY Cohort. Serum levels of the miRNAs were quantified by qRT-PCR in 682 women: 99 premenopausal and 583 postmenopausal women, with 1 and 122 women with prevalent fragility fractures in each group, respectively. We have collected clinical variables (such as age, prevalent, and incident fractures), bone turnover markers (BTMs), BMD by dual X-ray absorptiometry, and bone microarchitecture with HRpQCT. We observed a number of miRNAs to be associated with fragility fractures (prevalent or incident), BTMs, BMD, and microarchitecture. This effect, however, was negated after age adjustment. This may be because age was also strongly associated with the serum levels of the 32 miRNAs (correlation coefficient up to 0.49), confirming previous findings. In conclusion, in a well-characterized prospective cohort with a sizeable sample size, we found no evidence that these 32 preselected miRNAs were not associated with BTMs, BMD, microarchitecture, and or fragility fractures. © 2019 American Society for Bone and Mineral Research.

cited By 0; International audience; Periostin is an extracellular matrix protein that actively contributes to tumor progression and metastasis. Here, we hypothesized that it could be a marker of bone metastasis formation. To address this question, we used two polyclonal antibodies directed against the whole molecule or its C-terminal domain to explore the expression of intact and truncated forms of periostin in the serum and tissues (lung, heart, bone) of wild-type and periostin-deficient mice. In normal bones, periostin was expressed in the periosteum and specific periostin proteolytic fragments were found in bones, but not in soft tissues. In animals bearing osteolytic lesions caused by 4T1 cells, C-terminal intact periostin (iPTN) expression disappeared at the invasive front of skeletal tumors where bone-resorbing osteoclasts were present. In vitro, we found that periostin was a substrate for osteoclast-derived cathepsin K, generating proteolytic fragments that were not recognized by anti-periostin antibodies directed against iPTN. In vivo, using an in-house sandwich immunoassay aimed at detecting iPTN only, we observed a noticeable reduction of serum periostin levels (− 26%; P < 0.002) in animals bearing osteolytic lesions caused by 4T1 cells. On the contrary, this decrease was not observed in women with breast cancer and bone metastases when periostin was measured with a human assay detecting total periostin. Collectively, these data showed that mouse periostin was degraded at the bone metastatic sites, potentially by cathepsin K, and that the specific measurement of iPTN in serum should assist in detecting bone metastasis formation in breast cancer. © 2018, Springer Science+Business Media, LLC, part of Springer Nature.

The transcription factor TWIST1 induces epithelial-mesenchymal transition and/or escape to the oncogenic-induced failsafe program, facilitating the intravasation of breast cancer cells in the systemic circulation and their dissemination to the lungs. Its involvement in breast cancer bone metastasis is unknown. To address this question, human osteotropic MDA-MB-231/B02 breast cancer cells were stably transfected with a Tet-inducible vector encoding for TWIST1, whose expression was specifically repressed in the presence of doxycycline (dox). The intra-arterial inoculation of transfectants expressing TWIST1 in immunodeficient mice substantially increased the extent of osteolytic lesions in these animals, being 50% larger than that of animals bearing mock-transfected tumors, as determined by radiography. This difference was accompanied by a sharp reduction of the bone volume (indicating a higher bone destruction) and a twofold increase in the tumor volume compared with mice bearing mock-transfected tumors, as determined by histomorphometry. Importantly, the suppression of TWIST1 expression in MDA-MB-231/B02 cells in the presence of dox abolished the stimulatory effect of TWIST1 on bone metastasis formation in vivo. Additionally, examination of the bone marrow from untreated and dox-treated animals on day 7 after tumor cell inoculation, at which time there was no evidence of radiographic osteolytic lesions, revealed that the number of tumor cell colonies that were recovered from the bone marrow of untreated mice was dramatically increased compared with that of dox-fed animals. In vitro, TWIST1 expression promoted tumor cell invasion and enhanced microRNA 10b (miR-10b) expression, a proinvasive factor, but was dispensable for growth of tumor cells. In vivo, the repression of miR-10b substantially decreased the presence of TWIST1-expressing breast cancer cells in the bone marrow. Overall, these results establish that TWIST1 facilitates breast cancer bone metastasis formation through a mechanism dependent of miR-10b, which leads to increase tumor burden and bone destruction.

The development of bone metastases requires multistep and multicellular machinery consisting not only of processes shared with any type of metastases (formation of a pre-metastatic niche, chemotaxis of tumor cells into the host tissue, tumor cells escape from the microvasculature), but also biological interactions that are strictly related to the particular bone microenvironment (bone marrow colonization by cancer cells, osteomimicry, deregulation of bone homeostasis). MiRNAs are highly conserved, small RNAs molecules that regulate gene expression. The functional consequence of miRNA deregulation lies in the mRNA targets whose expression is altered. MiRNA networks acting as upstream regulators of these genes interfere with the initial steps of tumor local invasion and cancer cell intravasation, mainly by regulating the epithelial-mesenchymal transition, the motility, invasiveness and survival abilities of these cells. The miRNA-mediated regulation on the steps of bone tropism, anchorage, homing and finally bone colonization is more tissue specific, being dependent on the expression pattern of target miRNAs in bone marrow sinusoids, bone cells and microenvironment. In that, miRNA specific expression signatures that can distinguish between primary tumors from their corresponding bone metastases might be determinants of clinical aggressiveness. In this review, we focus on the current advances on functions and molecular mechanisms by which miRNAs exert their biological roles in regulating bone metastases development., info:eu-repo/semantics/published

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.

Lysyl oxidase (LOX) is a secreted copper-dependent amine oxidase whose primary function is to drive collagen crosslinking and extracellular matrix stiffness. LOX in colorectal cancer synergizes with hypoxia-inducible factor-1 (HIF-1) to promote tumor progression. Here we investigated whether LOX/HIF1 endows colorectal cancer cells with full competence for aggressive colonization in bone. We show that a high LOX expression in primary tumors from patients with colorectal cancer was associated with poor clinical outcome, irrespective of HIF-1. In addition, LOX was expressed by tumor cells in the bone marrow from colorectal cancer patients with bone metastases. In vivo experimental studies show that LOX overexpression in colorectal cancer cells or systemic delivery of the conditioned medium from LOX-overexpressing colorectal cancer cells promoted tumor cell dissemination in the bone marrow and enhanced osteolytic lesion formation, irrespective of HIF-1. Conversely, silencing or pharmacologic inhibition of LOX activity blocked dissemination of colorectal cancer cells in the bone marrow and tumor-driven osteolytic lesion formation. In vitro, tumor-secreted LOX supported the attachment and survival of colorectal cancer cells to and in the bone matrix, and inhibited osteoblast differentiation. LOX overexpression in colorectal cancer cells also induced a robust production of IL6. In turn, both LOX and IL6 were acting in concert to promote RANKL-dependent osteoclast differentiation, thereby creating an imbalance between bone resorption and bone formation. Collectively, our findings show that LOX supports colorectal cancer cell dissemination in the bone marrow and they reveal a novel mechanism through which LOX-driven IL6 production by colorectal cancer cells impairs bone homeostasis. Cancer Res; 77(2); 268–78. ©2016 AACR.

International audience; Bone metastases are one of the main complications of prostate cancer and they are incurable. We investigated whether and how estrogen receptor-related receptor alpha (ERRalpha) is involved in bone tumor progression associated with advanced prostate cancer. By meta-analysis, we first found that ERRalpha expression is correlated with castration-resistant prostate cancer (CRPC), the hallmark of progressive disease. We then analyzed tumor cell progression and the associated signaling pathways in gain-of-function/loss-of-function CRPC models in vivo and in vitro. Increased levels of ERRalpha in tumor cells led to rapid tumor progression, with both bone destruction and formation, and direct impacts on osteoclasts and osteoblasts. VEGF-A, WNT5A and TGFbeta1 were upregulated by ERRalpha in tumor cells and all of these factors also significantly and positively correlated withERRalpha expression in CRPC patient specimens. Finally, high levels of ERRalpha in tumor cells stimulated the pro-metastatic factor periostin expression in the stroma, suggesting that ERRalpha regulates the tumor stromal cell microenvironment to enhance tumor progression. Taken together, our data demonstrate that ERRalpha is a regulator of CRPC cell progression in bone. Therefore, inhibiting ERRalpha may constitute a new therapeutic strategy for prostate cancer skeletal-related events.

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.

Denosumab is a fully human monoclonal antibody to human RANKL, currently approved in the management of established bone metastases, mainly due to its anti-resorptive activity which blocks tumor-induced osteoclastogenesis and osteolysis. Our aims are to determine the role of RANK expression in breast cancer (BC) bone metastasis and the efficacy of RANKL blocking by AMG161 (IgG1 equivalent to Denosumab). AMG161 does not recognize murine RANKL, so we use a BalB/c nude knock-in (KI) mouse model, which expresses a chimeric murine/human RANKL. Moreover, non-invasive in vivo imaging techniques such as bioluminescence and µCT were used to monitor metastatic tumor burden and bone degradation, respectively. To evaluate bone marrow early colonization by BC cells, DiD labelling and ex vivo laser scanning multiphoton confocal microscopy were used. Long-term studies showed that intra-arterial (i.a.) injection of MDA-MB231 RANK versus MDA-MB231 leads to a poorer overall survival (OS), with the most striking differences seen in the earlier time-points. In this respect, the extent of radiographic osteolytic lesions was higher in the RANK overexpressing group than in the control group. Similar findings were observed following intra-tibial inoculation of RANK-expressing MCF-7 cells, when compared to that observed with parental MCF-7 cells. However, at later time points, bone and lung metastatic burden was similar between the two groups, suggesting that the poor OS of the RANK overexpressing group was due to a faster tumor progression owing to enhanced bone homing and colonization. Indeed, using short-term studies (5 and 14 days), i.a. injection of MDA-MB231 RANK led to higher incidence and extension of bone marrow micro-metastases than MDA-MB231, whereas there was no difference for lung micro-metastases. Daily subcutaneous injections of 1,5mg/kg AMG161 antibody to MDA-MB231RANK tumor-bearing animals, decreased bone micro-metastases and early bone marrow colonization without affecting lung micro-metastasis. We therefore hypothesized that RANK overexpressing BC cells interact with RANKL produced by osteocytes, osteoblasts and other cells in the osteogenic niche, favoring survival and colonization of BC cells in bone. To test this hypothesis, we made use of heterotypic mammosphere assays in which BC cells were cultured with or without cells of the osteogenic niche (e.g. pre-osteoblasts, mesenchymal stem cells), recombinant RANKL and anti-RANK antibody or AMG161. Co-culture with recombinant RANKL or a cellular source of RANKL increased BC cell mammosphere formation, and blocking RANK/RANKL signaling impaired it. In conclusion, RANK/RANKL signaling promotes the early engraftment of BC cells in the bone marrow. Blocking this signaling with AMG161 decreases bone marrow micro-metastasis formation in vivo, suggesting that adjuvant treatment of early-stage breast cancer patients with denosumab may prevent bone relapse. Citation Format: Sofia Sousa, Evelyne Gineyts, Sandra Geraci, Martine Croset, Philippe Clézardin. RANK-RANKL signaling inhibition delays early breast cancer bone metastasis formation [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 29.

Connexins (Cx) form gap junctions and allow direct cell-to-cell communication. Cx through gap junctions or by themselves play regulatory roles on cell growth and differentiation. Using genetically modified mice, we previously found that Cx32 acts as a down-regulator of growth in normal thyroid gland. In this study, we examined the impact of Cx32 ablation on oncogene-driven thyroid growth and neoplastic transformation. Cx32 knockout (Cx32-KO) mice were crossed with transgenic mice expressing, selectively in the thyroid gland, either the E7 or RET/PTC3 (RP3) oncogene. As already described, Cx32-KO mice had no detectable thyroid alteration in physiological conditions and mice expressing E7 or RP3 exhibited time-dependent thyroid hypertrophy and variable changes in expression of differentiation. The thyroid of E7 mice evolved towards a large colloid goitre whereas RP3 mice developed a hyperplastic thyroid of variable size, and the largest glands (about 40% of total) represented a profound tissue remodeling with proliferative papillary formations. E7-induced thyroid hypertrophy was reduced by about 40% in Cx32-KO mice as compared with wild-type (WT) littermates. On the contrary, thyroid hypertrophy induced by thyrotropin stimulation (in response to goitrogen treatment) was enhanced by about 40% in Cx32-KO mice as compared with WT mice. Thyroid hypertrophy of RP3 mice and the proportion of glands showing extensive tissue remodeling were drastically reduced in mice devoid of Cx32. Our data show that Cx32, which negatively controls thyroid growth activated by thyrotropin via the cAMP pathway, would act as a positive effector of thyroid growth triggered by oncogenes acting through other signaling cascades.

Atherosclerosis as a chronic inflammatory disease resulting from the imbalance of the pro- and anti-inflammatory factors in the vessel wall. PAF and PAF-like oxidized phospholipids generated upon LDL oxidation in the intima of the arteries may interact with infiltrated monocytes/macrophages and lead to the alteration of gene expression patterns accompanied by an impaired production of chemokines, interleukins and proteolytic and lipolytic enzymes. The aim of this study was to evaluate the binding capacity of the major component of PAF-like oxidized phospholipids, namely the 1-palmitoyl-2-oxovaleroyl-sn-glycero-3-phosphorylcholine (POVPC) to PAF-receptor (PAF-R) on the surface of human monocytes/macrophages and to further characterize the gene expression induced by such binding. We show that, POVPC binds to cultured human macrophages via PAF-R and transduces the signals leading to the intracellular Ca(2+) fluxes and modifies the transcription levels of numerous pro-inflammatory and pro-atherogenic genes. Although a some similarity of the gene expression patterns was observed when macrophages were activated with POVPC versus PAF, we observed that only POVPC treatment induced a several-fold activation of IL-8 gene. In turn, only PAF activated PAF-R, matrix metalloproteinase-13 and 15-lipoxygenase mRNA accumulation. Thus, we suggest, that POVPC signals in mature macrophages only in part through the PAF-R, a part of its effects may involve other receptors.

MicroRNAs (miRNAs) are highly conserved, small RNAs molecules that regulate gene expression. The functional consequence of miRNA deregulation in cancer and metastasis lies in the mRNA targets whose expression is altered. MiRNA networks acting as upstream regulators of these mRNAs interfere with the initial steps of tumor cell invasion and intravasation, mainly by regulating the epithelial-mesenchymal transition, the motility, invasiveness and survival abilities of cancer cells. The miRNA-mediated regulation of the steps associated with tumor cell tropism, anchorage, homing and colonization of the bone marrow is tissue-specific, being dependent on the regulation by miRNAs of a series of targeted mRNAs in bone marrow sinusoids, bone cells and the bone marrow microenvironment. In this respect, miRNA specific expression signatures that can distinguish between primary tumors and bone metastases might be clinically useful. The identification of metastatic bone-associated changes in miRNA expression suggests that miRNAs may be used as prognostic factors of bone relapse and that the manipulation of miRNA levels may represent a new therapeutic strategy to interfere with bone metastasis.

Skeletal metastases are complications of epithelial cancers, among which breast, prostate and lung carcinomas are the most osteotropic. In primary tumours, a subset of cancer cells undergoes epithelial-mesenchymal transition, acquires mobility to migrate into the surrounding stroma and seeds at distant sites to grow. The specific development of bone metastasis requires the recruitment of circulating tumour cells in the bone marrow, their adaptation to survive in the surrounding microenvironment where they alter the functions of osteoclasts and osteoblasts, and hijack signals coming from the bone matrix. Each of the molecular pathways underlining these steps is regulated by multiple factors, through the tight control of genes expressed by cancer cells interacting with cells from the bone microenvironment. In this context, miRNAs can act as master regulators of gene expression to control multiple aspects of bone metastasis formation, including cancer cell escape from the primary tumour site, cancer cell dissemination to bone and invasion of the bone marrow, as well as secondary outgrowth and tumour-stroma cell interactions. In the clinic, specific miRNA signatures have been identified in osteotropic cancer cells, raising the possibility that miRNAs could be used as biomarkers of bone metastasis. The regulatory activity of miRNAs in the bone microenvironment also suggests that miRNAs could be promising therapeutic targets.

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.

The transcription factor TWIST1 induces epithelial-mesenchymal transition and/or escape to the oncogenic-induced failsafe program, facilitating the intravasation of breast cancer cells in the systemic circulation and their dissemination to the lungs. Its involvement in breast cancer bone metastasis is unknown. To address this question, human osteotropic MDA-MB-231/B02 breast cancer cells were stably transfected with a Tet-inducible vector encoding for TWIST1, whose expression was specifically repressed in the presence of doxycycline (dox). The intra-arterial inoculation of transfectants expressing TWIST1 in immunodeficient mice substantially increased the extent of osteolytic lesions in these animals, being 50% larger than that of animals bearing mock-transfected tumors, as determined by radiography. This difference was accompanied by a sharp reduction of the bone volume (indicating a higher bone destruction) and a twofold increase in the tumor volume compared with mice bearing mock-transfected tumors, as determined by histomorphometry. Importantly, the suppression of TWIST1 expression in MDA-MB-231/B02 cells in the presence of dox abolished the stimulatory effect of TWIST1 on bone metastasis formation in vivo. Additionally, examination of the bone marrow from untreated and dox-treated animals on day 7 after tumor cell inoculation, at which time there was no evidence of radiographic osteolytic lesions, revealed that the number of tumor cell colonies that were recovered from the bone marrow of untreated mice was dramatically increased compared with that of dox-fed animals. In vitro, TWIST1 expression promoted tumor cell invasion and enhanced microRNA 10b (miR-10b) expression, a proinvasive factor, but was dispensable for growth of tumor cells. In vivo, the repression of miR-10b substantially decreased the presence of TWIST1-expressing breast cancer cells in the bone marrow. Overall, these results establish that TWIST1 facilitates breast cancer bone metastasis formation through a mechanism dependent of miR-10b, which leads to increase tumor burden and bone destruction.

A gas-chromatography combustion isotope ratio mass spectrometry (GCC-IRMS) method using carbon 13 (13C)-stable isotope to trace n-3 polyunsaturated fatty acids (PUFA) turnover in vivo is presented. Natural 13C abundance of commercial n-3 PUFA was measured from 100 to 300 ng of fatty acids and was −27.58, −27.83, and −28.16 for 22:6n-3, 22:5n-3, and 20:5n-3, expressed as δ13C‰ versus Pee Dee Belemnite (PDB), respectively. Precision of δ13C‰ values was comparable for the three PUFA and gave relative standard deviations of 0.95-0.97%. Isotope enrichment of 0.0010 at.% could be detected. Triglycerides enriched in [13C]22:6n-3 ([13C]22:6-TG) were synthesized by growing a microalgae on [l-13C]glucose. [13C]22:6n-3 represented 36 wt.% of total triglyceride fatty acids and had an isotope enrichment of 2.0420 at.%, which was the double of natural abundance. The isotope enrichment of 22:6n-3 in lipids from rat lipoproteins and red cells could be followed as a function of time after ingestion of 3 mg [13C]22:6-TG and showed specific patterns according to the lipid compartments. The retroconversion of [13C]22:6n-3 was also detected in HDL phosphatidylcholine by the appearance of [13C]22:5n-3 and [13C]20:5n-3. On the other hand, 22:6n-3 natural 13C abundance in human lipid classes of lipoproteins and blood cells has been measured using 10 ml plasma, even for the more limiting lipid compartments in terms of 22:6n-3 dose size. It is concluded that 13C-enriched n-3 PUFA along with GCC-IRMS can be used to study fatty acid fluxes in vivo, and that this method represents a convenient alternative to the utilization of radiotracer fatty acids in humans.

12(S)-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE), the end-lipoxygenase product of arachidonic acid in platelets has been previously shown to prevent PGH 2 /TXA 2 -induced aggregation. From the present study, we show that 12-HETE inhibits the binding of [ 125 I]-PTA-OH, a thromboxane antagonist, to platelet membranes with an IC 50 of 8 μM. This value is in accordance with previously reported 12-HETE concentrations required to prevent the aggregation induced by TxA 2 mimetics, the methano analogues of PGH 2 , U44069 and U46619. When [ 3 H]-U44069 was used as a thromboxane agonist to label intact platelets, 12-HETE also inhibited its binding. We conclude that part of the inhibitory effect of 12-HETE on PGH 2 /TxA 2 -induced aggregation might be the result of interacting with PGH 2 /TxA 2 receptor sites.

1. Benzodiazepine analogues inhibit human platelet aggregation induced by arachidonate with an EC50 value of 0.68 microM for PK 11195, the most potent analogue used. 2. There was a highly significant correlation between the inhibition of arachidonate-induced aggregation and the affinity for the peripheral-type of benzodiazepine binding sites. 3. There was no significant correlation between the inhibition of the platelet activating factor (PAF)-induced aggregation and the binding to the peripheral-type of benzodiazepine binding sites. 4. The inhibition of platelet aggregation seems to result from the inhibition of arachidonic acid cyclo-oxygenation, since the synthesis of thromboxane and 12-hydroxy-heptadecatrienoic acid, both cyclo-oxygenase products, was reduced. 5. Our results suggest that peripheral-type of benzodiazepine binding sites on human platelets could be linked to cyclo-oxygenase.

Elderly people present an increased incidence of atherosclerosis and vascular cerebral damages, associated with blood platelet hyperactivity and a stimulation of arachidonic acid metabolism in vivo. The effects of a low intake of purified eicosapentaenoic acid (EPA) on platelet hyperactivity in old human subjects has been investigated. In a randomized, double blind study, 8 people took during 2 months a daily intake of 100 mg of eicosapentaenoic acid (EPA) given as a triglyceride (1,3-dioctanoyl,2-eicosapentaenoyl-glycerol), and 8 other subjects ingested a placebo. A slight, but significant reduction of platelet-rich plasma aggregation in response to epinephrine and arachidonic acid occurred after EPA intake, as well as a decreased aggregation of washed platelets induced by thrombin, although collagen- and U-46619-induced aggregations were not significantly modified. EPA intake failed to affect arachidonic acid metabolism in thrombin-stimulated platelets or in clotted venous blood. The urinary excretion of thromboxane, 6-keto-PGF1 alpha and their 2,3-dinor-metabolites was also not modified. Similarly, no change in the plasma and platelet lipid fatty acid compositions could be observed. Platelet, but not plasma, alpha- and gamma-tocopherol were enhanced by EPA intake. An increase of platelet vitamin E has been associated with a decrease of aggregation, especially in vitamin E-deficient subjects, like elderly people. Therefore, low intake of EPA might have contributed to inhibit platelet aggregation by increasing cellular vitamin E.

Objective Argan oil is receiving increasing attention due to its potential health benefits in the prevention of cardiovascular risk, but no information to date is available about its possible effect on immune cells and functions. Methods To address this issue male rats were fed one of five diets that contained fish oil, argan oil, olive oil, coconut oil, or sunflower oil for 4 wk. The fatty acid composition of plasma and thymocyte lipids was then analyzed in relation to the mitogen-induced proliferation and phospholipase D (PLD) activity of thymocytes. Results The 18:2ω-6 proportion in thymocyte phospholipids from rats fed argan oil was significantly lower than that observed in phospholipids from rats fed sunflower oil and fish oil but higher than that found in the olive oil and coconut oil groups. Further, a significant positive linear relation was found between thymocyte proliferation and the 18:2ω-6 proportion in thymocyte phospholipids, whatever the diet. The proliferation response of thymocytes to mitogenic activation was also inversely correlated to PLD activity measured in intact thymocytes. Subsequent western blotting experiments indicated that the diet-induced variations in PLD activity mainly reflected variations in the expression of PLD2 protein. Conclusions On the whole, the present study shows that the effects of argan oil on immune cells are very similar to those of olive oil, and that, as a consequence, argan oil can be used as a balanced dietary supply without marked adverse effects on immune cell function.