Prevention Of Cancer Metastasis Research Articles

Metformin prevents cancer metastasis by inhibiting M2-like polarization of tumor associated macrophages.

Accumulated evidence suggests that M2-like polarized tumor associated macrophages (TAMs) plays an important role in cancer progression and metastasis, establishing TAMs, especially M2-like TAMs as an appealing target for therapy intervention. Here we found that metformin significantly suppressed IL-13 induced M2-like polarization of macrophages, as illustrated by reduced expression of CD206, down-regulation of M2 marker mRNAs, and inhibition of M2-like macrophages promoted migration of cancer cells and endothelial cells. Metformin triggered AMPKα1 activation in macrophage and silencing of AMPKα1 partially abrogated the inhibitory effect of metformin in IL-13 induced M2-like polarization. Administration of AICAR, another activator of AMPK, also blocked the M2-like polarization of macrophages. Metformin greatly reduced the number of metastases of Lewis lung cancer without affecting tumor growth. In tumor tissues, the percentage of M2-like macrophage was decreased and the area of pericyte-coated vessels was increased. Further, the anti-metastatic effect of metformin was abolished when the animals were treated with macrophages eliminating agent clodronate liposome. These findings suggest that metformin is able to block the M2-like polarization of macrophages partially through AMPKα1, which plays an important role in metformin inhibited metastasis of Lewis lung cancer.

Aspirin, lysine, mifepristone and doxycycline combined can effectively and safely prevent and treat cancer metastasis: prevent seeds from gemmating on soil.

Recent scientific advances have increased our understanding of the cancer metastatic complexities and provided further impetus for new combination therapies to prevent cancer metastasis. Here, we demonstrated that a combination (HAMPT) of aspirin, lysine, mifepristone and doxycycline can effectively and safely prevent cancer metastasis. The pharmaceutically-formulated HAMPT inhibited adhesion of cancer cells to either endothelial cells or extracellular matrix via down-regulating cell adhesion molecules ICAM-1 and α4-integrin. HAMPT inhibited the cloak effect by activated platelets on cancer cells, thereby interfering adhesion and invasion of cancer cells to the underlying stroma. At the effective concentration, HAMPT induced cancer cells into dormancy with minor inhibition on cell viability. Four-day pretreatment followed by 30-day oral administration of HAMPT (33.5-134 mg/kg) to the mice inoculated with cancer cells produced significant inhibition on cancer metastasis dose-dependently without marked side effects. Fifty-day rat toxicity study with HAMPT at doses (335-1340 mg/kg) 20-fold higher than its therapeutic dose produced no significant toxicity. Interestingly, the acute toxic death could not be reached at the maximum administrable dose (5 g/kg). This proof-of-concept study provides the first conceptual evidence that cancer metastasis can be controlled by using affordable old drugs to restrain circulating tumor cells from gemmating on the metastatic soil without the need for cytotoxicity.

Analysis of Chemokine Receptor Gene Expression in Esophageal Cancer Cells Compared with Breast Cancer with Insights into Metastasis.

Chemokine receptors have been shown to play an important role in the development and metastatic spread of various malignancies. In this study, the gene expression profile of some key chemokine receptors involved in metastasis has been investigated in esophageal and breast cancer cell lines. In a descriptive study, gene expression profile of CCR1, CCR6, CCR7, CCR9, CXCR1, and CXCR4 in human esophageal cancer cell line (KYSE-30) and human breast cancer cell line (MCF7) were analyzed using real-time PCR and their results were compared accordingly. We demonstrated for the first time the expression of CCR1, CCR6, CCR7, CCR9, CXCR1, and CXCR4 at transcriptional level in human esophageal cancer cell line. The expression of CCR1, CCR7 and CXCR4 were lower in esophageal compared with breast cancer cells, although without significant difference. CCR9 was highly expressed in esophageal cancer cells as compared to the breast cancer cells (P < 0.05). Similarly, the expression of CCR6 and CXCR1 were higher, although without significant difference. Esophageal cancer cells like breast cancer express some key chemokine receptors involved in metastasis. Targeting of proposed receptors in esophageal cancer may be a novel strategy for prevention of cancer metastasis.

The world's first clinical randomized trial of atrial natriuretic peptide for preventing cancer recurrence following lung cancer surgery

Most patients suffering from cancer die of metastatic disease. Surgical removal of solid tumors is performed as an initial attempt to cure patients if the primary tumor meets surgical indications. However, it is possible that surgical trauma itself influences the development of early recurrence. First, resection handling of the tumor can provoke detachment of tumor cells. Second, surgical trauma provokes the severe inflammatory reaction. Vascular inflammation is considered to render the endothelium adhesive to circulating tumor cells thereby allowing the metastasis of tumor cells. We have previously reported that administration of atrial natriuretic peptide (ANP) during the perioperative period reduces inflammatory response and has a prophylactic effect on postoperative cardiopulmonary complications in lung cancer surgery. Here we demonstrate that cancer recurrence after curative surgery was significantly lower in ANP-treated patients than in control patients (surgery alone). ANP is known to bind specifically to guanylyl cyclase-A (GC-A) receptor. In mouse models, we found that metastasis of GC-A–non-expressing tumor cells (i.e., B16 mouse melanoma cells) to the lung was increased in vascular endothelium-specificGC-A knockout mice and decreased in vascular endothelium-specific GC-A transgenic mice compared with control mice. ANP inhibited the adhesion of cancer cells to human pulmonary artery endothelial cells by suppressing the E-selectin expression that is promoted by inflammation. These results suggest that ANP prevents cancer metastasis by inhibiting the adhesion of tumor cells to inflamed endothelial cells. Thus, we have planned to start a multicenter randomized clinical trial to examine the use of perioperative administration of ANP for the prevention of cancer recurrence after lung cancer surgery in this year. In the near future, we want to expand the adaptation of ANP for various types of cancer surgery.

Apoptosis-induced cancer cell fusion: a mechanism of breast cancer metastasis.

Although cancer cell fusion has been suggested as a mechanism of cancer metastasis, the underlying mechanisms defining this process are poorly understood. In a recent study, apoptotic cells were newly identified as a type of cue that induces signaling via phosphatidylserine receptors to promote fusion of myoblasts. The microenvironment of breast tumors is often hypoxic, and because apoptosis is greatly increased in hypoxic conditions, we decided to investigate whether the mechanism of breast cancer cell fusion with mesenchymal stem/multipotent stromal cells (MSCs) involves apoptosis. We used a powerful tool for identification and tracking of hybrids based on bimolecular fluorescence complementation (BiFC) and found that breast cancer cells fused spontaneously with MSCs. This fusion was significantly enhanced with hypoxia and signaling associated with apoptotic cells, especially between nonmetastatic breast cancer cells and MSCs. In addition, the hybrids showed a significantly higher migratory capacity than did the parent cells. Taken together, these findings describe a mechanism by which hypoxia-induced apoptosis stimulates fusion between MSCs and breast tumor cells resulting in hybrids with an enhanced migratory capacity that may enable their dissemination to distant sites or metastases. In the long run, this study may provide new strategies for developing novel drugs for preventing cancer metastasis.

MiR-181a-5p Inhibits Cancer Cell Migration and Angiogenesis via Downregulation of Matrix Metalloproteinase-14.

Upregulation of matrix metalloproteinase MMP-14 (MT1-MMP) is associated with poor prognosis in cancer patients, but it is unclear how MMP-14 becomes elevated in tumors. Here, we show that miR-181a-5p is downregulated in aggressive human breast and colon cancers where its levels correlate inversely with MMP-14 expression. In clinical specimens, enhanced expression of MMP-14 was observed in cancer cells located at the invasive front of tumors where miR-181a-5p was downregulated relative to adjacent normal cells. Bioinformatics analyses defined a potential miR-181a-5p response element within the 3'-untranslated region of MMP-14 that was validated in reporter gene experiments. Ectopic miR-181a-5p reduced MMP-14 expression, whereas miR-181a-5p attenuation elevated MMP-14 expression. In support of a critical relationship between these two genes, miR-181a-5p-mediated reduction of MMP-14 levels was sufficient to decrease cancer cell migration, invasion, and activation of pro-MMP-2. Furthermore, this reduction in MMP-14 levels was sufficient to reduce in vivo invasion and angiogenesis in chick chorioallantoic membrane assays. Taken together, our results establish the regulation of MMP-14 in cancers by miR-181a-5p through a posttranscriptional mechanism, and they further suggest strategies to elevate miR-181a-5p to prevent cancer metastasis.

NDRG1: A Novel Therapeutic Target against Metastatic Cancers

Tumor metastasis is a critical clinical problem that requires urgent attention. Recently, there has been growing interest in the development of metastasis suppressor proteins as targets for anti-cancer therapy. N-myc downstream regulated protein 1 (NDRG1) is an important and well established metastasis suppressor protein that has shown promise as a significant therapeutic target to inhibit cancer metastasis. Notably, a novel series of thiosemicarbazone-based anti-cancer agents under development have shown the ability to up-regulate NDRG1. Therefore, further studies examining the effect of NDRG1 as a therapeutic target to prevent cancer metastasis are warranted.

Abstract BS2-2: Targeting the LIFR-Hippo-YAP pathway as an anti-metastatic strategy

Abstract Deregulation of signaling pathways that control organ size, such as the Hippo-YAP pathway, can lead to tumor formation and metastatic progression. Several upstream regulators are known to provide inputs feeding into the core Hippo pathway. However, the cell membrane receptors that activate the MST (the mammalian Hippo homolog)-LATS-YAP phosphorylation cascade are elusive. Although G-protein-coupled receptors have been found to regulate LATS and YAP phosphorylation, they act in a Hippo-independent manner and do not regulate MST. Combining high-throughput RNA sequencing, functional characterization, mechanistic studies, and clinical validation, we identified leukemia inhibitory factor receptor (LIFR) as a novel breast cancer metastasis suppressor downstream of the miR-9 microRNA and upstream of Hippo signaling. We found that LIFR suppresses metastasis by promoting cell membrane localization of the adaptor protein Scribble, leading to activation of a Hippo kinase cascade and functional inactivation of the transcriptional co-activator YAP. We found that LIFR is commonly downregulated in human breast cancer, and that loss of LIFR in non-metastatic stage I-III breast tumors was highly associated with poor metastasis-free, recurrence-free, and overall survival outcomes in approximately 1,000 patients. Recently, we generated Lifr conditional knockout (cKO) mice to determine whether genetic deletion of Lifr in the breast and other organs leads to increased organ size, tumorigenesis, and metastasis. Notably, we confirmed that Hippo signaling is shut down in Lifr-deficient adult mouse tissues and primary mouse embryonic fibroblasts. The phenotypic characterization of these mice is under way. In preliminary analyses, we indeed observed enlarged organs in Lifr cKO mice, which is what is expected from inactivation of Hippo signaling. These results further validated LIFR as a receptor that triggers the Hippo pathway. We aim to make translational advances on this finding. By delineating the appropriate cutoffs, detection of LIFR could be implemented in the clinic as a prognostic marker of metastatic relapse. We are also developing therapeutic agents to target the miR-9-LIFR-Hippo-YAP pathway as new anti-metastatic strategies. miR-9 antagomirs and LIFR ligands (such as LIF) could be useful for tumors with downregulation but not complete loss of LIFR, whereas agents targeting the functional YAP targets, such as CTGF, may be useful for tumors with either downregulation or complete loss of LIFR. Recently, we established collaboration with FibroGen to develop a human CTGF-neutralizing antibody (FG-3019) as an anti-metastatic agent in preclinical models of breast cancer. We will also investigate whether the blood level of CTGF can serve as a prognostic marker for metastatic relapse and a predictive marker to select patients that would benefit from FG-3019 treatment. These studies will likely facilitate biomarker-driven personalization of breast cancer metastasis prevention and treatment. Citation Format: Li Ma. Targeting the LIFR-Hippo-YAP pathway as an anti-metastatic strategy [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr BS2-2.

A pentacyclic triterpene natural product, ursolic acid and its prodrug US597 inhibit targets within cell adhesion pathway and prevent cancer metastasis.

Here we showed that ursolic acid (UA), a pentacyclic triterpene natural product, and its novel prodrug derivative US597 suppressed cancer cells adhesion, invasion and migration. This effect was accompanied by inhibition of focal adhesion signaling pathway including alterations in ICAM-1, VCAM-1, E-selectin, P-selectin, integrin α6β1, FAK, Src, paxillin and PTEN. While oral administration of UA or US597 increases survival rate of melanoma lung metastasis in C57BL/6 mice, US597 treatment extend the survival rate above that of UA. Immunohistochemical analysis revealed that US597 treatment regulates ICAM-1, a biomarker of metastasis. We did not detect side effects with US597 in mice such as weight loss, viscera tissues toxicity and blood cell abnormalities. Thus, UA and US597 are potential drug candidates for preventing cancer metastasis. Molecular and cellular study data suggest that UA and US597 modulate expression of cell adhesion molecules within focal adhesion signaling pathway leading to cancer cell motility.

RETRACTED: Atrial natriuretic peptide prevents cancer metastasis through vascular endothelial cells

Most patients suffering from cancer die of metastatic disease. Surgical removal of solid tumors is performed as an initial attempt to cure patients; however, surgery is often accompanied with trauma, which can promote early recurrence by provoking detachment of tumor cells into the blood stream or inducing systemic inflammation or both. We have previously reported that administration of atrial natriuretic peptide (ANP) during the perioperative period reduces inflammatory response and has a prophylactic effect on postoperative cardiopulmonary complications in lung cancer surgery. Here we demonstrate that cancer recurrence after curative surgery was significantly lower in ANP-treated patients than in control patients (surgery alone). ANP is known to bind specifically to NPR1 [also called guanylyl cyclase-A (GC-A) receptor]. In mouse models, we found that metastasis of GC-A-nonexpressing tumor cells (i.e., B16 mouse melanoma cells) to the lung was increased in vascular endothelium-specific GC-A knockout mice and decreased in vascular endothelium-specific GC-A transgenic mice compared with control mice. We examined the effect of ANP on tumor metastasis in mice treated with lipopolysaccharide, which mimics systemic inflammation induced by surgical stress. ANP inhibited the adhesion of cancer cells to pulmonary arterial and micro-vascular endothelial cells by suppressing the E-selectin expression that is promoted by inflammation. These results suggest that ANP prevents cancer metastasis by inhibiting the adhesion of tumor cells to inflamed endothelial cells.

Metformin, an old drug, brings a new era to cancer therapy.

Metformin is the most commonly prescribed drug for type 2 diabetes mellitus as it is inexpensive, safe, and efficient in ameliorating hyperglycemia and hyperinsulinemia. Epidemiological and clinical observation studies have shown that metformin use reduces risk of cancer in patients with type 2 diabetes mellitus and improves prognosis and survival rate of the cancer patients. Furthermore, ongoing clinical trials of metformin in cancer therapy are extending to nondiabetic population. Thus, metformin is emerging as an attractive candidate for cancer prevention and treatment. This review summarizes recent progress in studies of metformin use in cancer and discusses the possibility to enhance its efficacy and to prevent cancer metastasis.

Exploring cancer metastasis prevention strategy: interrupting adhesion of cancer cells to vascular endothelia of potential metastatic tissues by antibody-coated nanomaterial.

BackgroundCancer metastasis caused by circulating tumor cells (CTCs) accounts for 90% cancer-related death worldwide. Blocking the circulation of CTCs in bloodstream and their hetero-adhesion to vascular endothelia of the distant metastatic organs may prevent cancer metastasis. Nanomaterial-based intervention with adhesion between CTCs and endothelia has not been reported. Driven by the novel idea that multivalent conjugation of EpCAM and Slex antibodies to dendrimer surface may enhance the capacity and specificity of the nanomaterial conjugates for capturing and down-regulating colorectal CTCs, we conjugated the dendrimer nanomaterial with the EpCAM and Slex antibodies, and examined the capacity of the dual antibody-coated nanomaterial for their roles in interrupting CTCs-related cancer metastasis.ResultsThe antibody-coated nanomaterial was synthesized and characterized. The conjugates specifically bound and captured colon cancer cells SW620. The conjugate inhibited the cells’ viability and their adhesion to fibronectin (Fn)-coated substrate or human umbilical vein endothelial cells (HUVECs) in a concentration-dependent manner. In comparison with SW480 and LoVo cell lines, the activity and adhesion of SW620 to Fn-coated substrate and HUVECs were more specifically inhibited by the dual antibody conjugate because of the higher levels of EpCAM and Slex on SW620 cell surface. The hetero-adhesion between SW620 and Fn-coated substrate, or HUVECs was inhibited by about 60-70%. The dual conjugate showed the inhibition capacity more significant than its corresponding single antibody conjugates.ConclusionsThe present study provides the new evidence that coating nanomaterials with more than one antibody against CTCs may effectively interfere with the interaction between SW620 and HUVECs.Electronic supplementary materialThe online version of this article (doi:10.1186/s12951-015-0072-x) contains supplementary material, which is available to authorized users.

Enhanced Specificity in Capturing and Restraining Circulating Tumor Cells with Dual Antibody–Dendrimer Conjugates

Specifically capturing and restraining residual circulating tumor cells (CTCs) in cancer patients are the sine qua non for safely and effectively preventing cancer metastasis, to which the current chemotherapy has been limited due to its toxicity. Moreover, because of CTCs’ rarity and low activity, the current technology for capturing CTCs based solely on a single surface biomarker has limited capacity and is used mainly for in vitro diagnosis. Here, it is possible to sequentially conjugate two CTCs antibodies (aEpCAM and aSlex) to the functionalized dendrimers to specifically capture human hepatocellular CTCs in both artificial and clinical patient blood samples, and restrain their activities. The molecular entities of the conjugates are demonstrated by various means. The dual antibody conjugate captured CTCs threefold more than the single counterparts from the high concentrations of interfering red blood cells or leukocytes, as well as from the blood of liver cancer patients, and exhibits the superiority to their single counterparts in down‐regulating the captured CTCs. These results collectively provide the strong evidence that two antibodies can be compatibly conjugated to a nanomaterial, resulting in an enhanced specificity in restraining CTCs in blood.

Targeted delivery of a sialic acid-blocking glycomimetic to cancer cells inhibits metastatic spread.

Sialic acid sugars are overexpressed by cancer cells and contribute to the metastatic cascade at multiple levels. Therapeutic interference of sialic acids, however, has been difficult to pursue because of the absence of dedicated tools. Here we show that a rationally designed sialic acid-blocking glycomimetic (P-3F(ax)-Neu5Ac) successfully prevents cancer metastasis. Formulation of P-3F(ax)--Neu5Ac into poly(lactic-co-glycolic acid nanoparticles coated with antityrosinase-related protein-1 antibodies allowed targeted delivery of P-3F(ax)--Neu5Ac into melanoma cells, slow release, and long-term sialic acid blockade. Most importantly, intravenous injections of melanoma-targeting P-3F(ax)--Neu5Ac nanoparticles prevented metastasis formation in a murine lung metastasis model. These findings stress the importance of sialoglycans in cancer metastasis and advocate that sialic acid blockade using rationally designed glycomimetics targeted to cancer cells can effectively prevent cancer metastases. This targeting strategy to interfere with sialic acid-dependent processes is broadly applicable not only for different types of cancer but also in infection and inflammation.

Circulating tumor cells in oral squamous cell carcinoma: An insight.

Circulating tumor cells (CTCs) are those cells present in the blood and have antigenic and/or genetic characteristics of a specific tumor type. CTCs can be detected in the peripheral blood of cancer patients. Various techniques are available for detection of CTCs, which provide evidence for future metastasis. CTCs may provide new insight into the biology of cancer and process of metastasis in oral squamous cell carcinoma (OSCC). The detection of CTCs may represent a new diagnostic tool for predicting the occurrence of metastatic disease in OSCC and endow with the treatment strategies to efficiently treat and prevent cancer metastasis. This review gives an insight into the significance of CTCs and different techniques for detection of CTCs.

Galectin-3 as a Potential Target to Prevent Cancer Metastasis.

Interactions between two cells or between cell and extracellular matrix mediated by protein–carbohydrate interactions play pivotal roles in modulating various biological processes such as growth regulation, immune function, cancer metastasis, and apoptosis. Galectin-3, a member of the β-galactoside-binding lectin family, is involved in fibrosis as well as cancer progression and metastasis, but the detailed mechanisms of its functions remain elusive. This review discusses its structure, carbohydrate-binding properties, and involvement in various aspects of tumorigenesis and some potential carbohydrate ligands that are currently investigated to block galectin-3 activity.

Abstract B19: Brain metastasis depends on tumor cell initiated coagulation

Abstract Background: The incidence of brain metastasis exceeds that of primary brain tumors tenfold and is most frequently associated with lung cancer, breast cancer and melanoma. Despite a prognosis of only 6-9 months for patients with brain metastases, mechanisms of tumor cell brain colonization from the blood stream are unknown. Understanding this step could enable effective therapeutic strategies to prevent the development of incurable metastatic brain disease. We found that blood borne metastatic cancer cells reside within the cerebral microvasculature for several days and associate with platelets while crossing the blood brain barrier. Intravascular tumor cell survival and extravasation during this time may critically limit the success of brain metastasis. Activation of coagulation, platelets and fibrin formation contribute to tumor progression, cancer-associated thrombosis and metastatic spread to peripheral organs. However the function of coagulation in the seeding of brain metastases is unknown. Aims: We evaluated whether tumor cell-expressed tissue factor, a key activator of coagulation, promotes tumor cell seeding of brain metastases by initiating critical tumor cell-vascular interactions. Methods: We developed models of experimental brain metastasis to study how intravascular tumor cells cooperate with the coagulation system during early stages of brain colonization to survive and cross the blood brain barrier. Human breast cancer cells, injected into the left cardiac ventricle of immune deficient mice, are tracked during the initial phase of brain metastasis and progressive metastatic brain disease by detailed and quantitative histological analyses. We address how coagulation contributes to brain metastasis by targeting human tissue factor with specific inhibitory antibodies. We use ex-vivo bioluminescence imaging and immunohistochemical analyses at later stages of brain metastasis to determine the extent to which an early, transient treatment will result in long-term inhibition of brain metastatic disease. Results: We demonstrate that inhibition of tissue factor expressed by tumor cells reduces brain colonization. Targeting of tissue factor inhibits development of breast cancer brain metastasis and extends animal survival. We find that tissue factor properties which initiate coagulation or promote cytoprotective signaling pathways differentially contribute to brain metastasis development. Our findings indicate that the inhibition of coagulation can prevent the seeding of breast cancer cells into the brain. Conclusions: Our studies provide mechanistic insights into the process of tumor cell brain colonization and identify targets for development of therapeutics to prevent cancer metastasis to the brain and enhance patient survival. Citation Format: Laurie J. Gay, John Day, Sarah LeBoeuf, Melissa Ritland, Zaverio M. Ruggeri, Wolfram Ruf, Brunhilde H. Felding. Brain metastasis depends on tumor cell initiated coagulation. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr B19. doi:10.1158/1538-7445.CHTME14-B19

Role of pyruvate kinase M2 in transcriptional regulation leading to epithelial-mesenchymal transition.

Pyruvate kinase M2 (PKM2) is an alternatively spliced variant of the pyruvate kinase gene that is preferentially expressed during embryonic development and in cancer cells. PKM2 alters the final rate-limiting step of glycolysis, resulting in the cancer-specific Warburg effect (also referred to as aerobic glycolysis). Although previous reports suggest that PKM2 functions in nonmetabolic transcriptional regulation, its significance in cancer biology remains elusive. Here we report that stimulation of epithelial-mesenchymal transition (EMT) results in the nuclear translocation of PKM2 in colon cancer cells, which is pivotal in promoting EMT. Immunoprecipitation and LC-electrospray ionized TOF MS analyses revealed that EMT stimulation causes direct interaction of PKM2 in the nucleus with TGF-β-induced factor homeobox 2 (TGIF2), a transcriptional cofactor repressor of TGF-β signaling. The binding of PKM2 with TGIF2 recruits histone deacetylase 3 to the E-cadherin promoter sequence, with subsequent deacetylation of histone H3 and suppression of E-cadherin transcription. This previously unidentified finding of the molecular interaction of PKM2 in the nucleus sheds light on the significance of PKM2 expression in cancer cells.

Abstract 3293: Leptin induces breast cancer metastasis through a Neuropilin-1 (NRP-1)/OBR complex

Abstract Several studies have demonstrated Neuropilin-1 (NRP-1) implication in tumor progression independently of its known co-receptors. On the basis of growing connections between breast cancer and obesity, we postulated that leptin and its receptor OBR could be potential candidates. First, using NRP-1 over-expression in NRP-1 negative T47D or NRP-1 silencing in NRP-1 positive MDA-MB231 xenografts, we demonstrated that leptin induced metastasis in lymph node only in NRP-1 positive cells. Second, we demonstrated by co-immunoprecipitation, Proximity Ligation assay (duolink) and BRET analysis that NRP-1 is a co-receptor of OBR. Upon leptin exposure this complex is internalized to the nucleus. At this level, NRP-1 Chip-Seq analysis generated from MDA-MB-231 led to identify sequences of RNA polymerase II (Pol2) and transcription factor binding sites of genes implicated in breast cancer metastasis. This finding was confirmed by the detection of NRP-1/Pol2 interaction and by transcriptome analysis of RNA extracts from T47D-NRP-1 xenografts that shows modulation of these genes. In conclusion, we have demonstrated that NRP-1 is a co-receptor of OBR and upon leptin exposure may play a role as a transcription factor regulating genes involved in cancer progression. Thus, NRP-1 might be a therapeutic target to prevent cancer metastasis induced by leptin Citation Format: Zakia Belaid-Choucair, Julie Dam, Nicolas Goudin, Odile Filhole, Claude Cochet, Yves lepelletier, Tereza Coman, Geneviève Courtois, Alain Colige, Marie-Paule Defresne, Ralf Jockers, Olivier Hermine. Leptin induces breast cancer metastasis through a Neuropilin-1 (NRP-1)/OBR complex. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3293. doi:10.1158/1538-7445.AM2014-3293

Abstract 991: Brain metastasis depends on tumor cell initiated coagulation

Abstract Background: The incidence of brain metastasis exceeds that of primary brain tumors tenfold and is most frequently associated with lung cancer, breast cancer and melanoma. Despite a prognosis of only 6-9 months for patients with brain metastases, mechanisms of tumor cell brain colonization from the blood stream are unknown. Understanding this step could enable effective therapeutic strategies to prevent the development of incurable metastatic brain disease. We found that blood borne metastatic cancer cells reside within the cerebral microvasculature for several days and associate with platelets while crossing the blood brain barrier. Intravascular tumor cell survival and extravasation during this time may critically limit the success of brain metastasis. Activation of coagulation, platelets and fibrin formation contribute to tumor progression, cancer-associated thrombosis and metastatic spread to peripheral organs. However the function of coagulation in the seeding of brain metastases is unknown. Aims: We evaluated whether tumor cell-expressed tissue factor, a key activator of coagulation, promotes tumor cell seeding of brain metastases by initiating critical tumor cell-vascular interactions. Methods: We developed models of experimental brain metastasis to study how intravascular tumor cells cooperate with the coagulation system during early stages of brain colonization to survive and cross the blood brain barrier. Human breast cancer cells, injected into the left cardiac ventricle of immune deficient mice, are tracked during the initial phase of brain metastasis and progressive metastatic brain disease by detailed and quantitative histological analyses. We address how coagulation contributes to brain metastasis by targeting human tissue factor with specific inhibitory antibodies. We use ex-vivo bioluminescence imaging and immunohistochemical analyses at later stages of brain metastasis to determine the extent to which an early, transient treatment will result in long-term inhibition of brain metastatic disease. Results: We demonstrate that inhibition of tissue factor expressed by tumor cells reduces brain colonization. Targeting of tissue factor inhibits development of breast cancer brain metastasis and extends animal survival. We find that tissue factor properties which initiate coagulation or promote cytoprotective signaling pathways differentially contribute to brain metastasis development. Blockade of tissue factor-initiated coagulation inhibits intravascular tumor cell survival and prevents brain metastases in vivo. Our findings indicate that the inhibition of coagulation can prevent the seeding of breast cancer cells into the brain. Conclusions: Our studies provide mechanistic insights into the process of tumor cell brain colonization and identify targets for development of therapeutics to prevent cancer metastasis to the brain and enhance patient survival. Citation Format: Laurie J. Gay, John Day, Sarah LeBoeuf, Melissa Ritland, Zaverio Ruggeri, Wolfram Ruf, Brunhilde H. Felding. Brain metastasis depends on tumor cell initiated coagulation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 991. doi:10.1158/1538-7445.AM2014-991