Publications by authors named "Pierrick Fournier"

27 Publications

  • Page 1 of 1

Contribution of Macrophages and T Cells in Skeletal Metastasis.

Cancers (Basel) 2020 Apr 20;12(4). Epub 2020 Apr 20.

Biomedical Innovation Department, Centro de Investigación Científica y de Educación Superior de Ensenada, Ensenada, BC 22860, México.

Bone is a common site for metastases with a local microenvironment that is highly conducive for tumor establishment and growth. The bone marrow is replete with myeloid and lymphoid linage cells that provide a fertile niche for metastatic cancer cells promoting their survival and growth. Here, we discuss the role of macrophages and T cells in pro- and anti-tumoral mechanisms, their interaction to support cancer cell growth, and their contribution to the development of skeletal metastases. Importantly, immunotherapeutic strategies targeting macrophages and T cells in cancer are also discussed in this review as they represent a great promise for patients suffering from incurable bone metastases.
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http://dx.doi.org/10.3390/cancers12041014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7226332PMC
April 2020

TIE2 Induces Breast Cancer Cell Dormancy and Inhibits the Development of Osteolytic Bone Metastases.

Cancers (Basel) 2020 Apr 3;12(4). Epub 2020 Apr 3.

Biomedical Innovation Department, Centro de Investigación Científica y de Educación Superior de Ensenada (CICESE), Ensenada, Baja California 22860, Mexico.

Breast cancer (BCa) cells disseminating to the bone can remain dormant and resistant to treatments for many years until relapsing as bone metastases. The tyrosine kinase receptor TIE2 induces the dormancy of hematopoietic stem cells, and could also induce the dormancy of BCa cells. However, TIE2 is also a target for anti-angiogenic treatments in ongoing clinical trials, and its inhibition could then restart the proliferation of dormant BCa cells in bone. In this study, we used a combination of patient data, in vitro, and in vivo models to investigate the effect of TIE2 in the dormancy of bone metastases. In BCa patients, we found that a higher expression is associated with an increased time to metastases and survival. In vitro, TIE2 decreased cell proliferation as it increased the expression of cyclin-dependent kinase inhibitors and and arrested cells in the G/G phase. Expression of also increased the resistance to the chemotherapeutic 5-Fluorouracil. In mice, expression reduced tumor growth and the formation of osteolytic bone metastasis. Together, these results show that TIE2 is sufficient to induce dormancy in vitro and in vivo, and could be a useful prognostic marker for patients. Our data also suggest being cautious when using TIE2 inhibitors in the clinic, as they could awaken dormant disseminated tumor cells.
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http://dx.doi.org/10.3390/cancers12040868DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7226250PMC
April 2020

Brome mosaic virus-like particles as siRNA nanocarriers for biomedical purposes.

Beilstein J Nanotechnol 2020 20;11:372-382. Epub 2020 Feb 20.

Centro de Nanociencias y Nanotecnología - Universidad Nacional Autónoma de México (UNAM) - Ensenada, Baja California, México.

There is an increasing interest in the use of plant viruses as vehicles for anti-cancer therapy. In particular, the plant virus brome mosaic virus (BMV) and cowpea chlorotic mottle virus (CCMV) are novel potential nanocarriers for different therapies in nanomedicine. In this work, BMV and CCMV were loaded with a fluorophore and assayed on breast tumor cells. The viruses BMV and CCMV were internalized into breast tumor cells. Both viruses, BMV and CCMV, did not show cytotoxic effects on tumor cells in vitro. However, only BMV did not activate macrophages in vitro. This suggests that BMV is less immunogenic and may be a potential carrier for therapy delivery in tumor cells. Furthermore, BMV virus-like particles (VLPs) were efficiently loaded with small interfering RNA (siRNA) without packaging signal. The gene silencing was demonstrated by VLPs loaded with siGFP and tested on breast tumor cells that constitutively express the green fluorescent protein (GPF). After VLP-siGFP treatment, GFP expression was efficiently inhibited corroborating the cargo release inside tumor cells and the gene silencing. In addition, BMV VLP carring siAkt1 inhibited the tumor growth in mice. These results show the attractive potential of plant virus VLPs to deliver molecular therapy to tumor cells with low immunogenic response.
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http://dx.doi.org/10.3762/bjnano.11.28DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7059527PMC
February 2020

mutations increase variable new-antigen receptor single-domain antibodies for VEGF neutralization.

Oncotarget 2018 Jun 15;9(46):28016-28029. Epub 2018 Jun 15.

Departamento de Innovación Biomédica, Centro de Investigación Científica y de Educación Superior de Ensenada (CICESE), Ensenada, Baja California, México.

The stability, binding, and tissue penetration of variable new-antigen receptor (VNAR) single-domain antibodies have been tested as part of an investigation into their ability to serve as novel therapeutics. V13 is a VNAR that recognizes vascular endothelial growth factor 165 (VEGF). In the present study V13 was used as a parental molecule into which we introduced mutations designed . Two of the designed VNAR mutants were expressed, and their ability to recognize VEGF was assessed and . One mutation (Pro98Tyr) was designed to increase VEGF recognition, while the other (Arg97Ala) was designed to inhibit VEGF binding. Compared to parental V13, the Pro98Tyr mutant showed enhanced VEGF recognition and neutralization, as indicated by inhibition of angiogenesis and tumor growth. This molecule thus appears to have therapeutic potential for neutralizing VEGF in cancer treatment.
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http://dx.doi.org/10.18632/oncotarget.25549DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6021326PMC
June 2018

Functionalized rare earth-doped nanoparticles for breast cancer nanodiagnostic using fluorescence and CT imaging.

J Nanobiotechnology 2018 Mar 22;16(1):26. Epub 2018 Mar 22.

Biomedical Innovation Department, Centro de Investigación Científica y de Educación Superior de Ensenada (CICESE), Carretera Ensenada-Tijuana No. 3918, Zona Playitas, C.P. 22860, Ensenada, B.C., Mexico.

Background: Breast cancer is the second leading cause of cancer death among women and represents 14% of death in women around the world. The standard diagnosis method for breast tumor is mammography, which is often related with false-negative results leading to therapeutic delays and contributing indirectly to the development of metastasis. Therefore, the development of new tools that can detect breast cancer is an urgent need to reduce mortality in women. Here, we have developed GdO:Eu nanoparticles functionalized with folic acid (FA), for breast cancer detection.

Results: GdO:Eu nanoparticles were synthesized by sucrose assisted combustion synthesis and functionalized with FA using EDC-NHS coupling. The FA-conjugated GdO:Eu nanoparticles exhibit strong red emission at 613 nm with a quantum yield of ~ 35%. In vitro cytotoxicity studies demonstrated that the nanoparticles had a negligible cytotoxic effect on normal 293T and T-47D breast cancer cells. Cellular uptake analysis showed significantly higher internalization of FA-conjugated RE nanoparticles into T-47D cells (Folr ) compared to MDA-MB-231 breast cancer cells (Folr ). In vivo confocal and CT imaging studies indicated that FA-conjugated GdO:Eu nanoparticles accumulated more efficiently in T-47D tumor xenograft compared to the MDA-MB-231 tumor. Moreover, we found that FA-conjugated GdO:Eu nanoparticles were well tolerated at high doses (300 mg/kg) in CD1 mice after an intravenous injection. Thus, FA-conjugated GdO:Eu nanoparticles have great potential to detect breast cancer.

Conclusions: Our findings provide significant evidence that could permit the future clinical application of FA-conjugated GdO:Eu nanoparticles alone or in combination with the current detection methods to increase its sensitivity and precision.
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http://dx.doi.org/10.1186/s12951-018-0359-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5863469PMC
March 2018

Development of a functionalized UV-emitting nanocomposite for the treatment of cancer using indirect photodynamic therapy.

J Nanobiotechnology 2018 Feb 27;16(1):19. Epub 2018 Feb 27.

Biomedical Innovation Department, Centro de Investigación Científica y de Educación Superior de Ensenada, Baja California, (CICESE), Carretera Tijuana Ensenada No. 3918, Zona Playitas, 22860, Ensenada, Baja California, Mexico.

Background: Photodynamic therapy is a promising cancer therapy modality but its application for deep-seated tumor is mainly hindered by the shallow penetration of visible light. X-ray-mediated photodynamic therapy (PDT) has gained a major attention owing to the limitless penetration of X-rays. However, substantial outcomes have still not been achieved due to the low luminescence efficiency of scintillating nanoparticles and weak energy transfer to the photosensitizer. The present work describes the development of YPrAlO-based (YP) mesoporous silica coated nanoparticles, multifunctionalized with protoporphyrin IX (PpIX) and folic acid (YPMS@PpIX@FA) for potential application in targeted deep PDT.

Results: A YP nanophosphor core was synthesized using the sol-gel method to be used as X-ray energy transducer and was then covered with a mesoporous silica layer. The luminescence analysis indicated a good spectral overlap between the PpIX and nanoscintillator at the Soret as well as Q-band region. The comparison of the emission spectra with or without PpIX showed signs of energy transfer, a prerequisite for deep PDT. In vitro studies showed the preferential uptake of the nanocomposite in cancer cells expressing the folate receptorFolr1, validating the targeting efficiency. Direct activation of conjugated PpIX with UVA in vitro induced ROS production causing breast and prostate cancer cell death indicating that the PpIX retained its activity after conjugation to the nanocomposite. The in vivo toxicity analysis showed the good biocompatibility and non-immunogenic response of YPMS@PpIX@FA.

Conclusion: Our results indicate that YPMS@PpIX@FA nanocomposites are promising candidates for X-ray-mediated PDT of deep-seated tumors. The design of these nanoparticles allows the functionalization with exchangeable targeting ligands thus offering versatility, in order to target various cancer cells, expressing different molecular targets on their surface.
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http://dx.doi.org/10.1186/s12951-018-0344-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5827996PMC
February 2018

Halofuginone inhibits TGF-β/BMP signaling and in combination with zoledronic acid enhances inhibition of breast cancer bone metastasis.

Oncotarget 2017 Oct 23;8(49):86447-86462. Epub 2017 Sep 23.

Division of Endocrinology, Department of Medicine, Indiana University Purdue University at Indianapolis, Indiana, USA.

More efficient therapies that target multiple molecular mechanisms are needed for the treatment of incurable bone metastases. Halofuginone is a plant alkaloid-derivative with antiangiogenic and antiproliferative effects. Here we demonstrate that halofuginone is an effective therapy for the treatment of bone metastases, through multiple actions that include inhibition of TGFβ and BMP-signaling. Halofuginone blocked TGF-β-signaling in MDA-MB-231 and PC3 cells showed by inhibition of TGF-β-induced Smad-reporter, phosphorylation of Smad-proteins, and expression of TGF-β-regulated metastatic genes. Halofuginone increased inhibitory Smad7-mRNA and reduced TGF-β-receptor II protein. Proline supplementation but not Smad7-knockdown reversed halofuginone-inhibition of TGF-β-signaling. Halofuginone also decreased BMP-signaling. Treatment of MDA-MB-231 and PC3 cells with halofuginone reduced the BMP-Smad-reporter (BRE), Smad1/5/8-phosphorylation and mRNA of the BMP-regulated gene Id-1. Halofuginone decreased immunostaining of phospho-Smad2/3 and phospho-Smad1/5/8 in cancer cells . Furthermore, halofuginone decreased tumor-take and growth of orthotopic-tumors. Mice with breast or prostate bone metastases treated with halofuginone had significantly less osteolysis than control mice. Combined treatment with halofuginone and zoledronic-acid significantly reduced osteolytic area more than either treatment alone. Thus, halofuginone reduces breast and prostate cancer bone metastases in mice and combined with treatment currently approved by the FDA is an effective treatment for this devastating complication of breast and prostate-cancer.
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http://dx.doi.org/10.18632/oncotarget.21200DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5689697PMC
October 2017

The vitamin D receptor is involved in the regulation of human breast cancer cell growth via a ligand-independent function in cytoplasm.

Oncotarget 2017 Apr;8(16):26687-26701

Division of Endocrinology, Department of Medicine, Indiana University-Purdue University at Indianapolis, Indianapolis, Indiana, USA.

Vitamin D has pleiotropic effects on multiple tissues, including malignant tumors. Vitamin D inhibits breast cancer growth through activation of the vitamin D receptor (VDR) and via classical nuclear signaling pathways. Here, we demonstrate that the VDR can also function in the absence of its ligand to control behaviour of human breast cancer cells both outside and within the bone microenvironment. Stable shRNA expression was used to knock down VDR expression in MCF-7 cells, generating two VDR knockdown clonal lines. In ligand-free culture, knockdown of VDR in MCF-7 cells significantly reduced proliferation and increased apoptosis, suggesting that the VDR plays a ligand-independent role in cancer cell growth. Implantation of these VDR knockdown cells into the mammary fat pad of nude mice resulted in reduced tumor growth in vivo compared with controls. In the intra-tibial xenograft model, VDR knockdown greatly reduced the ability of the cells to form tumors in the bone microenvironment. The in vitro growth of VDR knockdown cells was rescued by the expression of a mutant form of VDR which is unable to translocate to the nucleus and hence accumulates in the cytoplasm. Thus, our data indicate that in the absence of ligand, the VDR promotes breast cancer growth both in vitro and in vivo and that cytoplasmic accumulation of VDR is sufficient to produce this effect in vitro. This new mechanism of VDR action in breast cancer cells contrasts the known anti-proliferative nuclear actions of the VDR-vitamin D ligand complex.
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http://dx.doi.org/10.18632/oncotarget.15803DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5432290PMC
April 2017

TGFβ-Mediated induction of SphK1 as a potential determinant in human MDA-MB-231 breast cancer cell bone metastasis.

Bonekey Rep 2015 8;4:719. Epub 2015 Jul 8.

Department of Pharmacology, Indiana University School of Medicine , Indianapolis, IN, USA ; Division of Endocrinology and Metabolism, Department of Medicine, Indiana University-Purdue University at Indianapolis , Indianapolis, IN, USA.

Mechanistic understanding of the preferential homing of circulating tumor cells to bone and their perturbation on bone metabolism within the tumor-bone microenvironment remains poorly understood. Alteration in both transforming growth factor β (TGFβ) signaling and sphingolipid metabolism results in the promotion of tumor growth and metastasis. Previous studies using MDA-MB-231 human breast cancer-derived cell lines of variable metastatic potential were queried for changes in sphingolipid metabolism genes to explore correlations between TGFβ dependence and bone metastatic behavior. Of these genes, only sphingosine kinase-1 (SPHK1) was identified to be significantly increased following TGFβ treatment. Induction of SPHK1 expression correlated to the degree of metastatic capacity in these MDA-MB-231-derived cell lines. We demonstrate that TGFβ mediates the regulation of SPHK1 gene expression, protein kinase activity and is critical to MDA-MB-231 cell viability. Furthermore, a bioinformatic analysis of human breast cancer gene expression supports SPHK1 as a hallmark TGFβ target gene that also bears the genetic fingerprint of the basal-like/triple-negative breast cancer molecular subtype. These data suggest a potential new signaling axis between TGFβ/SphK1 that may have a role in the development, prognosis or the clinical phenotype associated with tumor-bone metastasis.
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http://dx.doi.org/10.1038/bonekey.2015.88DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4495778PMC
July 2015

The TGF-β Signaling Regulator PMEPA1 Suppresses Prostate Cancer Metastases to Bone.

Cancer Cell 2015 Jun 14;27(6):809-21. Epub 2015 May 14.

Division of Endocrinology, Indiana University School of Medicine, Indianapolis, IN 46202, USA; Division of Endocrinology, University of Virginia School of Medicine, Charlottesville, VA 22903, USA. Electronic address:

Transforming growth factor-β (TGF-β) regulates the expression of genes supporting breast cancer cells in bone, but little is known about prostate cancer bone metastases and TGF-β. Our study reveals that the TGFBR1 inhibitor SD208 effectively reduces prostate cancer bone metastases. TGF-β upregulates in prostate cancer cells a set of genes associated with cancer aggressiveness and bone metastases, and the most upregulated gene was PMEPA1. In patients, PMEPA1 expression decreased in metastatic prostate cancer and low Pmepa1 correlated with decreased metastasis-free survival. Only membrane-anchored isoforms of PMEPA1 interacted with R-SMADs and ubiquitin ligases, blocking TGF-β signaling independently of the proteasome. Interrupting this negative feedback loop by PMEPA1 knockdown increased prometastatic gene expression and bone metastases in a mouse prostate cancer model.
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http://dx.doi.org/10.1016/j.ccell.2015.04.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4464909PMC
June 2015

FGF23 is elevated in multiple myeloma and increases heparanase expression by tumor cells.

Oncotarget 2015 Aug;6(23):19647-60

Department of Medicine, Indiana University School of Medicine, Indianapolis, IN, USA.

Multiply myeloma (MM) grows in and destroys bone, where osteocytes secrete FGF23, a hormone which affects phosphate homeostasis and aging. We report that multiple myeloma (MM) cells express receptors for and respond to FGF23. FGF23 increased mRNA for EGR1 and its target heparanase, a pro-osteolytic factor in MM. FGF23 signals through a complex of klotho and a classical FGF receptor (FGFR); both were expressed by MM cell lines and patient samples. Bone marrow plasma cells from 42 MM patients stained positively for klotho, while plasma cells from 8 patients with monoclonal gammopathy of undetermined significance (MGUS) and 6 controls were negative. Intact, active FGF23 was increased 2.9X in sera of MM patients compared to controls. FGF23 was not expressed by human MM cells, but co-culture with mouse bone increased its mRNA. The FGFR inhibitor NVP-BGJ398 blocked the heparanase response to FGF23. NVP-BGJ398 did not inhibit 8226 growth in vitro but significantly suppressed growth in bone and induction of the osteoclast regulator RANK ligand, while decreasing heparanase mRNA. The bone microenvironment provides resistance to some anti-tumor drugs but increased the activity of NVP-BGJ398 against 8226 cells. The FGF23/klotho/heparanase signaling axis may offer targets for treatment of MM in bone.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4637311PMC
http://dx.doi.org/10.18632/oncotarget.3794DOI Listing
August 2015

Halofuginone inhibits the establishment and progression of melanoma bone metastases.

Cancer Res 2012 Dec 20;72(23):6247-56. Epub 2012 Sep 20.

Division of Endocrinology and Metabolism, Department of Medicine, Indiana University-Purdue University Indianapolis, Walther Hall, C132 980 W Walnut Street, Indianapolis, IN 46202, USA.

TGF-β derived from bone fuels melanoma bone metastases by inducing tumor secretion of prometastatic factors that act on bone cells to change the skeletal microenvironment. Halofuginone is a plant alkaloid derivative that blocks TGF-β signaling with antiangiogenic and antiproliferative properties. Here, we show for the first time that halofuginone therapy decreases development and progression of bone metastasis caused by melanoma cells through the inhibition of TGF-β signaling. Halofuginone treatment of human melanoma cells inhibited cell proliferation, phosphorylation of SMAD proteins in response to TGF-β, and TGF-β-induced SMAD-driven transcription. In addition, halofuginone reduced expression of TGF-β target genes that enhance bone metastases, including PTHrP, CTGF, CXCR4, and IL11. Also, cell apoptosis was increased in response to halofuginone. In nude mice inoculated with 1205 Lu melanoma cells, a preventive protocol with halofuginone inhibited bone metastasis. The beneficial effects of halofuginone treatment were comparable with those observed with other anti-TGF-β strategies, including systemic administration of SD208, a small-molecule inhibitor of TGF-β receptor I kinase, or forced overexpression of Smad7, a negative regulator of TGF-β signaling. Furthermore, mice with established bone metastases treated with halofuginone had significantly less osteolysis than mice receiving placebo assessed by radiography. Thus, halofuginone is also effective in reducing the progression of melanoma bone metastases. Moreover, halofuginone treatment reduced melanoma metastasis to the brain, showing the potential of this novel treatment against cancer metastasis.
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http://dx.doi.org/10.1158/0008-5472.CAN-12-1444DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4447239PMC
December 2012

TGF-beta-RI kinase inhibitor SD-208 reduces the development and progression of melanoma bone metastases.

Cancer Res 2011 Jan 16;71(1):175-84. Epub 2010 Nov 16.

Division of Endocrinology, Department of Medicine, Indiana University PurdueUniversity at Indianapolis, Indianapolis, USA.

Melanoma often metastasizes to bone where it is exposed to high concentrations of TGF-β. Constitutive Smad signaling occurs in human melanoma. Because TGF-β promotes metastases to bone by several types of solid tumors including breast cancer, we hypothesized that pharmacologic blockade of the TGF-β signaling pathway may interfere with the capacity of melanoma cells to metastasize to bone. In this study, we tested the effect of a small molecule inhibitor of TGF-β receptor I kinase (TβRI), SD-208, on various parameters affecting the development and progression of melanoma, both in vitro and in a mouse model of human melanoma bone metastasis. In melanoma cell lines, SD-208 blocked TGF-β induction of Smad3 phosphorylation, Smad3/4-specific transcription, Matrigel invasion and expression of the TGF-β target genes PTHrP, IL-11, CTGF, and RUNX2. To assess effects of SD-208 on melanoma development and metastasis, nude mice were inoculated with 1205Lu melanoma cells into the left cardiac ventricle and drug was administered by oral gavage on prevention or treatment protocols. SD-208 (60 mg/kg/d), started 2 days before tumor inoculation prevented the development of osteolytic bone metastases compared with vehicle. In mice with established bone metastases, the size of osteolytic lesions was significantly reduced after 4 weeks treatment with SD-208 compared with vehicle-treated mice. Our results demonstrate that therapeutic targeting of TGF-β may prevent the development of melanoma bone metastases and decrease the progression of established osteolytic lesions.
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http://dx.doi.org/10.1158/0008-5472.CAN-10-2651DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3225124PMC
January 2011

Nitrogen-containing bisphosphonates can inhibit angiogenesis in vivo without the involvement of farnesyl pyrophosphate synthase.

Bone 2011 Feb 20;48(2):259-66. Epub 2010 Oct 20.

Institut National de la Santé et de la Recherche Médicale (INSERM), UMR 664, IFR62, F-69372 Lyon, France.

Nitrogen-containing bisphosphonates (N-BPs) are widely used to block bone destruction associated with bone metastasis because they are effective inhibitors of osteoclast-mediated bone resorption. More specifically, once internalized by osteoclasts, N-BPs block the activity of farnesyl pyrophosphate synthase (FPPS), a key enzyme in the mevalonate pathway. In addition to their antiresorptive activity, preclinical evidence shows that N-BPs have antiangiogenic properties. However, the exact reasons for which N-BPs inhibit angiogenesis remain largely unknown. Using different angiogenesis models, we examined here the effects of zoledronate, risedronate and three structural analogs of risedronate (NE-58025, NE-58051 and NE-10790) with lower potencies to inhibit FPPS activity. Risedronate and zoledronate were much more potent than NE-compounds at inhibiting both endothelial cell proliferation in vitro and vessel sprouting in the chicken egg chorioallantoic membrane (CAM) assay. In addition, only risedronate and zoledronate inhibited the revascularization of the prostate gland in testosterone-stimulated castrated rats. Moreover, as opposed to NE-compounds, risedronate and zoledronate induced intracellular accumulation of isopentenyl pyrophosphate (IPP) in endothelial cells by blocking the activity of the IPP-consuming enzyme FPPS. Thus, these results indicated that N-BPs inhibited angiogenesis in a FPPS-dependent manner. However, drug concentrations used to inhibit angiogenesis, both in vitro and in the CAM and prostate gland assays, were high. In contrast, a low concentration of risedronate (1 μM) was sufficient to inhibit blood vessel formation in the ex vivo rat aortic ring assay. Moreover, NE-58025 (which had a 7-fold lower potency than risedronate to inhibit FPPS activity) was as effective as risedronate to reduce angiogenesis in the rat aortic ring assay. In conclusion, our results suggest that low concentrations of N-BPs inhibit angiogenesis in a FPPS-independent manner, whereas higher drug concentrations were required to inhibit FPPS activity in vivo.
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http://dx.doi.org/10.1016/j.bone.2010.09.035DOI Listing
February 2011

GLI2-mediated melanoma invasion and metastasis.

J Natl Cancer Inst 2010 Aug 21;102(15):1148-59. Epub 2010 Jul 21.

Curie Institute, INSERM U1021/CNRS UMR3347, University Center, Orsay Cedex, France.

Background: The transforming growth factor-beta (TGF-beta) pathway, which has both tumor suppressor and pro-oncogenic activities, is often constitutively active in melanoma and is a marker of poor prognosis. Recently, we identified GLI2, a mediator of the hedgehog pathway, as a transcriptional target of TGF-beta signaling.

Methods: We used real-time reverse transcription-polymerase chain reaction (RT-PCR) and western blotting to determine GLI2 expression in human melanoma cell lines and subsequently classified them as GLI2high or as GLI2low according to their relative GLI2 mRNA and protein expression levels. GLI2 expression was reduced in a GLI2high cell line with lentiviral expression of short hairpin RNA targeting GLI2. We assessed the role of GLI2 in melanoma cell invasiveness in Matrigel assays. We measured secretion of matrix metalloproteinase (MMP)-2 and MMP-9 by gelatin zymography and expression of E-cadherin by western blotting and RT-PCR. The role of GLI2 in development of bone metastases was determined following intracardiac injection of melanoma cells in immunocompromised mice (n = 5-13). Human melanoma samples (n = 79) at various stages of disease progression were analyzed for GLI2 and E-cadherin expression by immunohistochemistry, in situ hybridization, or RT-PCR. All statistical tests were two-sided.

Results: Among melanoma cell lines, increased GLI2 expression was associated with loss of E-cadherin expression and with increased capacity to invade Matrigel and to form bone metastases in mice (mean osteolytic tumor area: GLI2high vs GLI2low, 2.81 vs 0.93 mm(2), difference = 1.88 mm(2), 95% confidence interval [CI] = 1.16 to 2.60, P < .001). Reduction of GLI2 expression in melanoma cells that had expressed high levels of GLI2 substantially inhibited both basal and TGF-beta-induced cell migration, invasion (mean number of Matrigel invading cells: shGLI2 vs shCtrl (control), 52.6 vs 100, difference = 47.4, 95% CI = 37.0 to 57.8, P = .024; for shGLI2 + TGF-beta vs shCtrl + TGF-beta, 31.0 vs 161.9, difference = -130.9, 95% CI = -96.2 to -165.5, P = .002), and MMP secretion in vitro and the development of experimental bone metastases in mice. Within human melanoma lesions, GLI2 expression was heterogeneous, associated with tumor regions in which E-cadherin was lost and increased in the most aggressive tumors.

Conclusion: GLI2 was directly involved in driving melanoma invasion and metastasis in this preclinical study.
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http://dx.doi.org/10.1093/jnci/djq257DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2914763PMC
August 2010

How do bisphosphonates inhibit bone metastasis in vivo?

Neoplasia 2010 Jul;12(7):571-8

Institut National de la Santé et de la Recherche Médicale, UMR 664, IFR62, Lyon, France.

Bisphosphonates are potent inhibitors of osteoclast-mediated bone resorption and have demonstrated clinical utility in the treatment of patients with osteolytic bone metastases. They also exhibit direct antitumor activity in vitro and can reduce skeletal tumor burden and inhibit the formation of bone metastases in vivo. However, whether such effects are caused by a direct action of bisphosphonates on tumor cells or indirectly through inhibition of bone resorption remains unclear. To address this question, we used here a structural analog of the bisphosphonate risedronate, NE-58051, which has a bone mineral affinity similar to that of risedronate, but a 3000-fold lower bone antiresorptive activity. In vitro, risedronate and NE-58051 inhibited proliferation of breast cancer and melanoma cell lines. In vivo, risedronate and NE-58051 did not inhibit the growth of subcutaneous B02 breast tumor xenografts or the formation of B16F10 melanoma lung metastasis. In contrast to NE-58051, risedronate did inhibit B02 breast cancer bone metastasis formation by reducing both bone destruction and skeletal tumor burden, indicating that the antitumor effect of bisphosphonates is achieved mainly through inhibition of osteoclast-mediated bone resorption.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2907583PMC
http://dx.doi.org/10.1593/neo.10282DOI Listing
July 2010

Hypoxia and TGF-beta drive breast cancer bone metastases through parallel signaling pathways in tumor cells and the bone microenvironment.

PLoS One 2009 Sep 3;4(9):e6896. Epub 2009 Sep 3.

Division of Endocrinology, Department of Medicine, University of Virginia, Charlottesville, Virginia, United States of America.

Background: Most patients with advanced breast cancer develop bone metastases, which cause pain, hypercalcemia, fractures, nerve compression and paralysis. Chemotherapy causes further bone loss, and bone-specific treatments are only palliative. Multiple tumor-secreted factors act on the bone microenvironment to drive a feed-forward cycle of tumor growth. Effective treatment requires inhibiting upstream regulators of groups of prometastatic factors. Two central regulators are hypoxia and transforming growth factor (TGF)- beta. We asked whether hypoxia (via HIF-1alpha) and TGF-beta signaling promote bone metastases independently or synergistically, and we tested molecular versus pharmacological inhibition strategies in an animal model.

Methodology/principal Findings: We analyzed interactions between HIF-1alpha and TGF-beta pathways in MDA-MB-231 breast cancer cells. Only vascular endothelial growth factor (VEGF) and the CXC chemokine receptor 4 (CXCR4), of 16 genes tested, were additively increased by both TGF-beta and hypoxia, with effects on the proximal promoters. We inhibited HIF-1alpha and TGF-beta pathways in tumor cells by shRNA and dominant negative receptor approaches. Inhibition of either pathway decreased bone metastasis, with no further effect of double blockade. We tested pharmacologic inhibitors of the pathways, which target both the tumor and the bone microenvironment. Unlike molecular blockade, combined drug treatment decreased bone metastases more than either alone, with effects on bone to decrease osteoclastic bone resorption and increase osteoblast activity, in addition to actions on tumor cells.

Conclusions/significance: Hypoxia and TGF-beta signaling in parallel drive tumor bone metastases and regulate a common set of tumor genes. In contrast, small molecule inhibitors, by acting on both tumor cells and the bone microenvironment, additively decrease tumor burden, while improving skeletal quality. Our studies suggest that inhibitors of HIF-1alpha and TGF-beta may improve treatment of bone metastases and increase survival.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0006896PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2731927PMC
September 2009

Agents targeting prostate cancer bone metastasis.

Anticancer Agents Med Chem 2009 Dec;9(10):1079-88

Department of Medicine, Division of Endocrinology, Indiana University, Indianapolis, Indiana 46202, USA.

Bone is the most common site for metastasis of advanced prostate cancers. Once housed in the skeleton, tumors are incurable and cause protracted morbidity, and bone metastases may contribute to mortality through unknown mechanisms. Bone provides a unique microenvironment whose local interactions with tumor cells offer novel targets for therapeutic interventions. Many standard cancer treatments cause bone loss, which may aggravate skeletal metastases, although this is preventable with approved agents. Improved bone-targeted treatments can decrease the serious skeletal morbidities associated with metastatic prostate cancer and may in the future improve overall survival. The development of such treatments requires preclinical evaluation in animal models of prostate cancer growth in bone.
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http://dx.doi.org/10.2174/187152009789735008DOI Listing
December 2009

Lowering bone mineral affinity of bisphosphonates as a therapeutic strategy to optimize skeletal tumor growth inhibition in vivo.

Cancer Res 2008 Nov;68(21):8945-53

Faculté de Médecine Laennec, Institut National de la Santé et de la Recherche Médicale, Research Unit UMR 664, Lyon, France.

Bisphosphonates bind avidly to bone mineral and are potent inhibitors of osteoclast-mediated bone destruction. They also exhibit antitumor activity in vitro. Here, we used a mouse model of human breast cancer bone metastasis to examine the effects of risedronate and NE-10790, a phosphonocarboxylate analogue of the bisphosphonate risedronate, on osteolysis and tumor growth. Osteolysis was measured by radiography and histomorphometry. Tumor burden was measured by fluorescence imaging and histomorphometry. NE-10790 had a 70-fold lower bone mineral affinity compared with risedronate. It was 7-fold and 8,800-fold less potent than risedronate at reducing, respectively, breast cancer cell viability in vitro and bone loss in ovariectomized animals. We next showed that risedronate given at a low dosage in animals bearing human B02-GFP breast tumors reduced osteolysis by inhibiting bone resorption, whereas therapy with higher doses also inhibited skeletal tumor burden. Conversely, therapy with NE-10790 substantially reduced skeletal tumor growth at a dosage that did not inhibit osteolysis, a higher dosage being able to also reduce bone destruction. The in vivo antitumor activity of NE-10790 was restricted to bone because it did not inhibit the growth of subcutaneous B02-GFP tumor xenografts nor the formation of B16-F10 melanoma lung metastases. Moreover, NE-10790, in combination with risedronate, reduced both osteolysis and skeletal tumor burden, whereas NE-10790 or risedronate alone only decreased either tumor burden or osteolysis, respectively. In conclusion, our study shows that decreasing the bone mineral affinity of bisphosphonates is an effective therapeutic strategy to inhibit skeletal tumor growth in vivo.
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http://dx.doi.org/10.1158/0008-5472.CAN-08-2195DOI Listing
November 2008

Molecular biology of bone metastasis.

Mol Cancer Ther 2007 Oct;6(10):2609-17

University of Virginia Department of Medicine, Division of Endocrinology, Charlottesville, Virginia, USA.

Metastasis is a final stage of tumor progression. Breast and prostate cancer cells preferentially metastasize to bone, wherein they cause incurable osteolytic and osteoblastic lesions. The bone matrix is rich in factors, such as transforming growth factor-beta and insulin-like growth factors, which are released into the tumor microenvironment by osteolysis. These factors stimulate the growth of tumor cells and alter their phenotype, thus promoting a vicious cycle of metastasis and bone pathology. Physical factors within the bone microenvironment, including low oxygen levels, acidic pH, and high extracellular calcium concentrations, may also enhance tumor growth. These elements of the microenvironment are potential targets for chemotherapeutic intervention to halt tumor growth and suppress bone metastasis.
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http://dx.doi.org/10.1158/1535-7163.MCT-07-0234DOI Listing
October 2007

BMP7: a new bone metastases prevention?

Am J Pathol 2007 Sep 9;171(3):739-43. Epub 2007 Aug 9.

Department of Internal Medicine, Division of Endocrinology and Metabolism, University of Virginia, PO Box 801419, Charlottesville, VA 22908-1419, USA.

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http://dx.doi.org/10.2353/ajpath.2007.070582DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1959486PMC
September 2007

Stable overexpression of Smad7 in human melanoma cells impairs bone metastasis.

Cancer Res 2007 Mar;67(5):2317-24

INSERM U697, Hôpital Saint-Louis, 1 Avenue Claude Vellefaux, 75010 Paris, France.

Melanoma has a propensity to metastasize to bone, where it is exposed to high concentrations of transforming growth factor-beta (TGF-beta). Because TGF-beta promotes bone metastases from other solid tumors, such as breast cancer, we tested the role of TGF-beta in melanoma metastases to bone. 1205Lu melanoma cells, stably transfected to overexpress the natural TGF-beta/Smad signaling inhibitor Smad7, were studied in an experimental model of bone metastasis whereby tumor cells are inoculated into the left cardiac ventricle of nude mice. All mice bearing parental and mock-transfected 1205Lu cells developed osteolytic bone metastases 5 weeks post-tumor inoculation. Mice bearing 1205Lu-Smad7 tumors had significantly less osteolysis on radiographs and longer survival compared with parental and mock-transfected 1205Lu mice. To determine if the reduced bone metastases observed in mice bearing 1205Lu-Smad7 clones was due to reduced expression of TGF-beta target genes known to enhance metastases to bone from breast cancer cells, we analyzed gene expression of osteolytic factors, parathyroid hormone-related protein (PTHrP) and interleukin-11 (IL-11), the chemotactic receptor CXCR4, and osteopontin in 1205Lu cells. Quantitative reverse transcription-PCR analysis indicated that PTHrP, IL-11, CXCR4, and osteopontin mRNA steady-state levels were robustly increased in response to TGF-beta and that Smad7 and the TbetaRI small-molecule inhibitor, SB431542, prevented such induction. In addition, 1205Lu-Smad7 bone metastases expressed significantly lower levels of IL-11, connective tissue growth factor, and PTHrP. These data suggest that TGF-beta promotes osteolytic bone metastases due to melanoma by stimulating the expression of prometastatic factors via the Smad pathway. Blockade of TGF-beta signaling may be an effective treatment for melanoma metastasis to bone.
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http://dx.doi.org/10.1158/0008-5472.CAN-06-3950DOI Listing
March 2007

New insights into the role of T cells in the vicious cycle of bone metastases.

Curr Opin Rheumatol 2006 Jul;18(4):396-404

Division of Endocrinology and Metabolism, Department of Medicine, University of Virginia, Charlottesville, Virginia 22908-1419, USA.

Purpose Of Review: Bone metastases interact with the bone microenvironment. Cancer cells modulate the functions of osteoblasts and osteoclasts to induce new bone formation or bone resorption, leading to secondary stimulation of tumor development. Recent findings suggest the involvement of T cells in this process.

Recent Findings: Bone metastatic cancer cells produce factors such as parathyroid hormone-related protein, interleukin-7, and interleukin-8 that can recruit or activate T cells. T cells are involved in bone remodeling and can induce osteoclastic resorption. Bone resorption releases transforming growth factor-beta, however, which could suppress T-cell antitumor immune responses. Bisphosphonate antiresorptive drugs are the approved treatment for solid tumor bone metastases. They have recently been found to activate the cytolytic activity of gammadelta T cells. Thus, inhibitors of transforming growth factor-beta or antiresorptive therapies may be effective enhancers of antitumor immune responses in bone.

Summary: T cells at the site of bone metastases may be functionally suppressed by factors in the bone microenvironment. Instead of acting against tumor cells, they may increase bone resorption, making bone a privileged site for tumor growth.
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http://dx.doi.org/10.1097/01.bor.0000231909.35043.daDOI Listing
July 2006

Bisphosphonates and cancer-induced bone disease: beyond their antiresorptive activity.

Cancer Res 2005 Jun;65(12):4971-4

Institut National de la Sante et de la Recherche Medicale Research Unit 664, Laennec School of Medicine, Lyon, France.

Bisphosphonates are primarily known for their ability to inhibit osteoclast-mediated bone resorption. They are an indispensable part of therapy for patients with cancers that cause osteolysis. However, there is now a growing body of evidence from preclinical research showing that bisphosphonates also exhibit antitumor activity, both in vitro and in vivo. They can affect molecular mechanisms of tumor cell adhesion, invasion, and proliferation; reinforce the effects of cytotoxic agents in a synergistic manner; and exhibit antiangiogenic and immunomodulatory effects. These preclinical findings reveal exciting ways of optimizing bisphosphonate therapy in oncology to fully exploit their antitumor potential.
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http://dx.doi.org/10.1158/0008-5472.CAN-05-0264DOI Listing
June 2005

Angiostatin inhibits bone metastasis formation in nude mice through a direct anti-osteoclastic activity.

J Biol Chem 2003 Nov 3;278(46):45826-32. Epub 2003 Sep 3.

INSERM Research Unit 403, Faculté de Médecine Laënnec, 69372 Lyon Cedex 08, France.

Bone is a very common metastatic site for breast cancer. In bone metastasis, there is a vicious circle wherein bone-residing metastatic cells stimulate osteoclast-mediated bone resorption, and bone-derived growth factors released from resorbed bone promote tumor growth. The contribution of tumor angiogenesis in the growth of bone metastases is, however, unknown. By using an experimental model of bone metastasis caused by MDA-MB-231/B02 breast cancer cells that quite closely mimics the conditions likely to occur in naturally arising metastatic human breast cancers, we demonstrate here that when MDA-MB-231/B02 cells were engineered to produce at the bone metastatic site an angiogenesis inhibitor, angiostatin, there was a marked inhibition in the extent of skeletal lesions. Inhibition of skeletal lesions came with a pronounced reduction in tumor burden in bone. However, although angiostatin produced by MDA-MB-231/B02 cells was effective at inhibiting in vitro endothelial cell proliferation and in vivo angiogenesis in a Matrigel implant model, we have shown that it inhibited cancer-induced bone destruction through a direct inhibition of osteoclast activity and generation. Overall, these results indicate that, besides its well known anti-angiogenic activity, angiostatin must also be considered as a very effective inhibitor of bone resorption, broadening its potential clinical use in cancer therapy.
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http://dx.doi.org/10.1074/jbc.M309024200DOI Listing
November 2003

In vitro and in vivo antitumor effects of bisphosphonates.

Curr Med Chem 2003 Jan;10(2):173-80

INSERM Research Unit 403, Faculty of Medicine Laënnec, Lyon, France.

Bisphosphonates are powerful inhibitors of osteoclast-mediated bone resorption. They are currently used in the palliative treatment of bone metastases. However, bisphosphonates do not only act on osteoclasts. There is now extensive in vitro preclinical evidence that bisphosphonates can act on tumor cells: they inhibit tumor cell adhesion to mineralized bone as well as tumor cell invasion and proliferation. Bisphosphonates induce also tumor cell apoptosis and stimulate gammadelta T cell cytotoxicity against tumor cells. In vivo, bisphosphonates inhibit bone metastasis formation and reduce skeletal tumor burden. This may reflect direct antitumor effects and indirect effects via inhibition of bone resorption. In addition, bisphosphonates inhibit experimental angiogenesis in vitro and in vivo. Understanding the molecular mechanisms through which bisphosphonates act on tumor and endothelial cells will be undoubtedly an important task in the future. It will allow the design of clinical trials to investigate whether the antitumor activity of bisphosphonates can be realized in the clinical setting.
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http://dx.doi.org/10.2174/0929867033368529DOI Listing
January 2003

Bisphosphonates inhibit angiogenesis in vitro and testosterone-stimulated vascular regrowth in the ventral prostate in castrated rats.

Cancer Res 2002 Nov;62(22):6538-44

Institut National de la Santé et de la Recherche Médicale, Research Unit 403, Faculty of Medicine Laënnec, Lyon 69372, France.

Bisphosphonates (BPs) are used currently in the treatment of patients with bone metastases because these compounds inhibit bone resorption. We examined here the effects of BPs on inhibition of endothelial cell functions in vitro and in vivo. Treatment of endothelial cells with BPs (clodronate, risedronate, ibandronate, and zoledronic acid) reduced proliferation, induced apoptosis, and decreased capillary-like tube formation in vitro. Quantification of blood vessels in bone biopsy specimens from patients with Paget's disease before and after clodronate treatment showed a 40% reduction of the vascularization after BP treatment. However, such a decreased vascularity could be secondary to a reduction of bone resorption. Therefore, the tissue distribution of [14C]BPs in male rats was examined to develop an angiogenesis model in a noncalcified tissue where BPs could accumulate. [14C]BPs (zoledronic acid, ibandronate, and clodronate) not only accumulated in bone but also transiently accumulated in the prostate. The effects of BPs on testosterone-induced revascularization of the prostate gland in castrated rats were then studied. Testosterone in combination with ibandronate or zoledronic acid induced a 17-35% reduction of the prostate weight compared with castrated rats treated with testosterone alone. Blood vessel immunostaining on prostate tissue sections revealed that both ibandronate and zoledronic acid induced a 50% reduction of the revascularization of the prostate gland. Moreover, zoledronic acid did not alter testosterone-induced activity of a luciferase gene reporter construct transfected in androgen-dependent prostatic cells, indicating that this BP did not directly interfere with testosterone. In conclusion, BPs have in vivo antiangiogenic properties, which could be of relevance to improve therapy and prevention of bone metastasis. In addition, our results extend the potential clinical use of BPs to patients with early prostate cancer.
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November 2002