Marion David

Cancer Cell Expression of Autotaxin Controls Bone Metastasis Formation in Mouse through Lysophosphatidic Acid-Dependent Activation of Osteoclasts

Background Bone metastases are highly frequent complications of breast cancers. Current bone metastasis treatments using powerful anti-resorbtive agents are only palliative indicating that factors independent of bone resorption control bone metastasis progression. Autotaxin (ATX/NPP2) is a secreted protein with both oncogenic and pro-metastatic properties. Through its lysosphospholipase D (lysoPLD) activity, ATX controls the level of lysophosphatidic acid (LPA) in the blood. Platelet-derived LPA promotes the progression of osteolytic bone metastases of breast cancer cells. We asked whether ATX was involved in the bone metastasis process. We characterized the role of ATX in osteolytic bone metastasis formation by using genetically modified breast cancer cells exploited on different osteolytic bone metastasis mouse models. Methodology/Principal Findings Intravenous injection of human breast cancer MDA-B02 cells with forced expression of ATX (MDA-B02/ATX) to inmmunodeficiency BALB/C nude mice enhanced osteolytic bone metastasis formation, as judged by increased bone loss, tumor burden, and a higher number of active osteoclasts at the metastatic site. Mouse breast cancer 4T1 cells induced the formation of osteolytic bone metastases after intracardiac injection in immunocompetent BALB/C mice. These cells expressed active ATX and silencing ATX expression inhibited the extent of osteolytic bone lesions and decreased the number of active osteoclasts at the bone metastatic site. In vitro, osteoclast differentiation was enhanced in presence of MDA-B02/ATX cell conditioned media or recombinant autotaxin that was blocked by the autotaxin inhibitor vpc8a202. In vitro, addition of LPA to active charcoal-treated serum restored the capacity of the serum to support RANK-L/MCSF-induced osteoclastogenesis. Conclusion/Significance Expression of autotaxin by cancer cells controls osteolytic bone metastasis formation. This work demonstrates a new role for LPA as a factor that stimulates directly cancer growth and metastasis, and osteoclast differentiation. Therefore, targeting the autotaxin/LPA track emerges as a potential new therapeutic approach to improve the outcome of patients with bone metastases.

Interaction of platelet-derived autotaxin with tumor integrin αVβ3 controls metastasis of breast cancer cells to bone.

Autotaxin (ATX), through its lysophospholipase D activity controls physiological levels of lysophosphatidic acid (LPA) in blood. ATX is overexpressed in multiple types of cancers, and together with LPA generated during platelet activation promotes skeletal metastasis of breast cancer. However, the pathophysiological sequelae of regulated interactions between circulating LPA, ATX, and platelets remain undefined in cancer. In this study, we show that ATX is stored in α-granules of resting human platelets and released upon tumor cell-induced platelet aggregation, leading to the production of LPA. Our in vitro and in vivo experiments using human breast cancer cells that do not express ATX (MDA-MB-231 and MDA-B02) demonstrate that nontumoral ATX controls the early stage of bone colonization by tumor cells. Moreover, expression of a dominant negative integrin αvβ3-Δ744 or treatment with the anti-human αvβ3 monoclonal antibody LM609, completely abolished binding of ATX to tumor cells, demonstrating the requirement of a fully active integrin αvβ3 in this process. The present results establish a new mechanism for platelet contribution to LPA-dependent metastasis of breast cancer cells, and demonstrate the therapeutic potential of disrupting the binding of nontumor-derived ATX with the tumor cells for the prevention of metastasis.

Chemical optimization of new ligands of the low-density lipoprotein receptor as potential vectors for central nervous system targeting.

Drug delivery to the central nervous system is hindered by the presence of physiological barriers such as the blood-brain barrier. To accomplish the task of nutrient transport, the brain endothelium is endowed with various transport systems, including receptor-mediated transcytosis (RMT). This system can be used to shuttle therapeutics into the central nervous system (CNS) in a noninvasive manner. Therefore, the low-density lipoprotein receptor (LDLR) is a relevant target for delivering drugs. From an initial phage display biopanning, a series of peptide ligands for the LDLR was optimized leading to size reduction and improved receptor binding affinity with the identification of peptide 22 and its analogues. Further real-time biphoton microscopy experiments on living mice demonstrated the ability of peptide 22 to efficiently and quickly cross CNS physiological barriers. This validation of peptide 22 led us to explore its binding on the extracellular LDLR domain from an NMR-oriented structural study and docking experiments.

LRP-1–CD44, a New Cell Surface Complex Regulating Tumor Cell Adhesion

ABSTRACT The low-density lipoprotein receptor-related protein 1 (LRP-1) is a large endocytic receptor mediating the clearance of various molecules from the extracellular matrix. In the field of cancer, LRP-1-mediated endocytosis was first associated with antitumor properties. However, recent results suggested that LRP-1 may coordinate the adhesion-deadhesion balance in malignant cells to support tumor progression. Here, we observed that LRP-1 silencing or RAP (receptor-associated protein) treatment led to accumulation of CD44 at the tumor cell surface. Moreover, we evidenced a tight interaction between CD44 and LRP-1, not exclusively localized in lipid rafts. Overexpression of LRP-1-derived minireceptors indicated that the fourth ligand-binding cluster of LRP-1 is required to bind CD44. Labeling of CD44 with EEA1 and LAMP-1 showed that internalized CD44 is routed through early endosomes toward lysosomes in a LRP-1-dependent pathway. LRP-1-mediated internalization of CD44 was highly reduced under hyperosmotic conditions but poorly affected by membrane cholesterol depletion, revealing that it proceeds mostly via clathrin-coated pits. Finally, we demonstrated that CD44 silencing abolishes RAP-induced tumor cell attachment, revealing that cell surface accumulation of CD44 under LRP-1 blockade is mainly responsible for the stimulation of tumor cell adhesion. Altogether, our data shed light on the LRP-1-mediated internalization of CD44 that appeared critical to define the adhesive properties of tumor cells.

Proteasome inhibitors induce FLT3-ITD degradation through autophagy in AML cells

Internal tandem duplication of the Fms-like tyrosine kinase-3 receptor (FLT3) internal tandem duplication (ITD) is found in 30% of acute myeloid leukemia (AML) and is associated with a poor outcome. In addition to tyrosine kinase inhibitors, therapeutic strategies that modulate the expression of FLT3-ITD are also promising. We show that AML samples bearing FLT3-ITD mutations are more sensitive to proteasome inhibitors than wild-type samples and this sensitivity is strongly correlated with a higher FLT3-ITD allelic burden. Using pharmacologic inhibitors of autophagy, specific downregulation of key autophagy proteins including Vps34, autophagy gene (Atg)5, Atg12, Atg13, biochemical, and microscopy studies, we demonstrated that proteasome inhibitors induced cytotoxic autophagy in AML cells. FLT3-ITD molecules were detectable within autophagosomes after bortezomib treatment indicating that autophagy induction was responsible for the early degradation of FLT3-ITD, which preceded the inhibition of mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK), PI3K/AKT, and STAT5 pathways, and subsequent activation of cell death. Moreover, proteasome inhibitors overcome resistance to quizartinib induced by mutations in the kinase domain of FLT3, suggesting that these compounds may prevent the emergence of mutant clones arising from tyrosine kinase inhibitor treatments. In xenograft mice models, bortezomib stimulated the conversion of LC3-I to LC3-II, indicating induction of autophagy in vivo, downregulated FLT3-ITD protein expression and improved overall survival. Therefore, selecting patients according to FLT3-ITD mutations could be a new way to detect a significant clinical activity of proteasome inhibitors in AML patients.

Targeting lysophosphatidic acid receptor type 1 with Debio 0719 inhibits spontaneous metastasis dissemination of breast cancer cells independently of cell proliferation and angiogenesis

Metastasis is the main cause of death for cancer patients. Targeting factors that control metastasis formation is a major challenge for clinicians. Lysophosphatidic acid (LPA) is a bioactive phospholipid involved in cancer. LPA activates at least six independent G protein-coupled receptors (LPA1–6). Tumor cells frequently co-express multiple LPA receptors, puzzling the contribution of each one to cancer progression. All three receptors, LPA1, LPA2 and LPA3, act as oncogenes and prometastatic factors in the mouse mammary gland. The competitive inhibitor of LPA1 and LPA3 receptors, Ki16425, inhibits efficiently breast cancer bone metastases in animal models. We showed here that Debio 0719, which corresponds to the R-stereoisomer of Ki16425 exhibited highest antagonist activities at LPA1 (IC50=60 nM) and LPA3 (IC50=660 nM) than Ki16425 [IC50=130 nM (LPA1); IC50=2.3 μM (LPA3)]. In vitro, Debio 0719, inhibited LPA-dependent invasion of the 4T1 mouse mammary cancer cells. In vivo, early but not late administration of Debio 0719 (50 mg/kg p.o. twice daily) to BALB/c mice during the course of orthotopic 4T1 primary tumor growth reduced the number of spontaneously disseminated tumor cells to bone and lungs without affecting the growth of primary tumors and tumor-induced angiogenesis. We found that increased LPA1 mRNA expression in primary tumors of breast cancer patients correlated significantly with their positive lymph node status (p<0.001). Altogether, our results suggest that LPA1 controls early events of metastasis independently of cell proliferation and angiogenesis. Therefore, targeting this receptor with Debio 0719 has a high therapeutic potential against metastasis formation for breast cancer patients.

Pleiotropic activity of lysophosphatidic acid in bone metastasis.

Bone is a common metastatic site for solid cancers. Bone homeostasis is tightly regulated by intimate cross-talks between osteoblast (bone forming cells) and osteoclasts (bone resorbing cells). Once in the bone microenvironment, metastatic cells do not alter bone directly but instead perturb the physiological balance of the bone remodeling process controlled by bone cells. Tumor cells produce growth factors and cytokines stimulating either osteoclast activity leading to osteolytic lesions or osteoblast function resulting in osteoblastic metastases. Growth factors, released from the resorbed bone matrix or throughout osteoblastic bone formation, sustain tumor growth. Therefore, bone metastases are the sites of vicious cycles wherein tumor growth and bone metabolism sustain each other. Lysophosphatidic acid (LPA) promotes the growth of primary tumors and metastatic dissemination of cancer cells. We have shown that by acting on cancer cells via the contribution of blood platelets and the LPA-producing enzyme Autotaxin (ATX), LPA promotes the progression of osteolytic bone metastases in animal models. In the light of recent reports it would appear that the role of LPA in the context of bone metastases is complex involving multiple sources of lipid combined with direct and indirect effects on target cells. This review will present our current knowledge on the LPA/ATX axis involvement in osteolytic and osteoblastic skeletal metastases and will discuss the potential activity of LPA upstream and downstream metastasis seeding of cancer cells to bone as well as its implication in cancer induced bone pain. This article is part of a Special Issue entitled Advances in Lysophospholipid Research.

Characterization of a Sphingosine 1-Phosphate Receptor Antagonist Prodrug

Sphingosine 1-phosphate (S1P) is a phospholipid that binds to a set of G protein-coupled receptors (S1P1–S1P5) to initiate an array of signaling cascades that affect cell survival, differentiation, proliferation, and migration. On a larger physiological scale, the effects of S1P on immune cell trafficking, vascular barrier integrity, angiogenesis, and heart rate have also been observed. An impetus for the characterization of S1P-initiated signaling effects came with the discovery that FTY720 [fingolimod; 2-amino-2-(2-[4-octylphenyl]ethyl)-1,3-propanediol] modulates the immune system by acting as an agonist at S1P1. In the course of structure-activity relationship studies to better understand the functional chemical space around FTY720, we discovered conformationally constrained FTY720 analogs that behave as S1P receptor type-selective antagonists. Here, we present a pharmacological profile of a lead S1P1/3 antagonist prodrug, 1-(hydroxymethyl)-3-(3-octylphenyl)cyclobutane (VPC03090). VPC03090 is phosphorylated by sphingosine kinase 2 to form the competitive antagonist species 3-(3-octylphenyl)-1-(phosphonooxymethyl)cyclobutane (VPC03090-P) as observed in guanosine 5′-O-(3-[35S]thio)triphosphate binding assays, with effects on downstream S1P receptor signaling confirmed by Western blot and calcium mobilization assays. Oral dosing of VPC03090 results in an approximate 1:1 phosphorylated/alcohol species ratio with a half-life of 30 h in mice. Because aberrant S1P signaling has been implicated in carcinogenesis, we applied VPC03090 in an immunocompetent mouse mammary cancer model to assess its antineoplastic potential. Treatment with VPC03090 significantly inhibited the growth of 4T1 primary tumors in mice. This result calls to attention the value of S1P receptor antagonists as not only research tools but also potential therapeutic agents.

Identification of heparin-binding EGF-like growth factor (HB-EGF) as a biomarker for lysophosphatidic acid receptor type 1 (LPA1) activation in human breast and prostate cancers.

Lysophosphatidic acid (LPA) is a natural bioactive lipid with growth factor-like functions due to activation of a series of six G protein-coupled receptors (LPA1–6). LPA receptor type 1 (LPA1) signaling influences the pathophysiology of many diseases including cancer, obesity, rheumatoid arthritis, as well as lung, liver and kidney fibrosis. Therefore, LPA1 is an attractive therapeutic target. However, most mammalian cells co-express multiple LPA receptors whose co-activation impairs the validation of target inhibition in patients because of missing LPA receptor-specific biomarkers. LPA1 is known to induce IL-6 and IL-8 secretion, as also do LPA2 and LPA3. In this work, we first determined the LPA induced early-gene expression profile in three unrelated human cancer cell lines expressing different patterns of LPA receptors (PC3: LPA1,2,3,6; MDA-MB-231: LPA1,2; MCF-7: LPA2,6). Among the set of genes upregulated by LPA only in LPA1-expressing cells, we validated by QPCR and ELISA that upregulation of heparin-binding EGF-like growth factor (HB-EGF) was inhibited by LPA1–3 antagonists (Ki16425, Debio0719). Upregulation and downregulation of HB-EGF mRNA was confirmed in vitro in human MDA-B02 breast cancer cells stably overexpressing LPA1 (MDA-B02/LPA1) and downregulated for LPA1 (MDA-B02/shLPA1), respectively. At a clinical level, we quantified the expression of LPA1 and HB-EGF by QPCR in primary tumors of a cohort of 234 breast cancer patients and found a significantly higher expression of HB-EGF in breast tumors expressing high levels of LPA1. We also generated human xenograph prostate tumors in mice injected with PC3 cells and found that a five-day treatment with Ki16425 significantly decreased both HB-EGF mRNA expression at the primary tumor site and circulating human HB-EGF concentrations in serum. All together our results demonstrate that HB-EGF is a new and relevant biomarker with potentially high value in quantifying LPA1 activation state in patients receiving anti-LPA1 therapies.

Use of LDL receptor–targeting peptide vectors for in vitro and in vivo cargo transport across the blood-brain barrier

The blood‐brain barrier (BBB) prevents the entry of many drugs into the brain and, thus, is a major obstacle in the treatment of CNS diseases. There is some evidence that the LDL receptor (LDLR) is expressed at the BBB and may participate in the transport of endogenous ligands from blood to brain, a process referred to as receptor‐mediated transcytosis. We previously described a family of peptide vectors that were developed to target the LDLR. In the present study, in vitro BBB models that were derived from wild‐type and LDLR‐knockout animals (ldlr‒/‒) were used to validate the specific LDLR‐dependent transcytosis of LDL via a nondegradative route. We next showed that LDLR‐targeting peptide vectors, whether in fusion or chemically conjugated to an Ab Fc fragment, promote binding to apical LDLR and transendothelial transfer of the Fc fragment across BBB monolayers via the same route as LDL. Finally, we demonstrated in vivo that LDLR significantly contributes to the brain uptake of vectorized Fc. We thus provide further evidence that LDLR is a relevant receptor for CNS drug delivery via receptor‐mediated transcytosis and that the peptide vectors we developed have the potential to transport drugs, including proteins or Ab based, across the BBB.—Molino, Y., David, M., Varini, K., Jabès, F., Gaudin, N., Fortoul, A., Bakloul, K., Masse, M., Bernard, A., Drobecq, L., Lécorché, P., Temsamani, J., Jacquot, G., Khrestchatisky, M. Use of LDL receptor–targeting peptide vectors for in vitro and in vivo cargo transport across the blood‐brain barrier. FASEB J. 31, 1807–1827 (2017). www.fasebj.org