Experimental Metastasis Mouse Model Research Articles

Abstract A111: Plant galactolipid dLGG suppresses lung metastasis of melanoma through modulating endothelial-mesenchymal transition extravasation and oxylipins dynamics

Abstract A galactolipid 1,2-di-O-linolenoyl-3-O-ß-galactopyranosyl-sn-glycerol (designated dLGG) isolated from medicinal plant Crassocephalum rabens (Asteraceae) was demonstrated to be anti-inflammatory and anti-melanoma tumor growth in syngeneic mouse system in our laboratory. In this study, an experimental lung metastasis mouse model using a B16 melanoma cell clone (designated B16COX-2/Luc) carrying COX-2 promoter driven luciferase reporter gene was established to monitor the cancer cell growth and metastasis in animals and the dLGG effect. Our data showed that dLGG (20 mg/kg BW, dLGG−20), cisplatin (2 mg/kg BW, CP−2), and combination drug treatment (CP−2+dLGG−20) significantly inhibited lung metastasis of melanoma in mice from 100% at tumor control group to 9.2%, 43.1%, and 4.1%, respectively, as determined by bioluminescence intensity. The dLGG (30±8 days), cisplatin (35±9 days) and combination treatment group (29±7 days) mice showed longer mean survival time than tumor control group (22±1 days)(P<0.02). Immunohistochemistry study on lung tissues showed that dLGG and CP+dLGG combination treatment inhibited the expression of cell proliferation (PCNA), cell invasion (MMP2 and MMP9), and cell metastasis (COX-2, CXCR4 and CD9) markers, but less effective in cisplatin group. We investigated the underlying mechanism of dLGG on inhibiting melanoma cell activity, including endothelial-mesenchymal transition (EMT), and extravasation through tight junction permeability. Our data showed that EMT markers expression, such as effector vimentin, regulator snail, and inducer TGF-ß, ß-catenin and TNF-α were down-regulated in dLGG-treated melanoma cells. Melanoma culture medium (CM) induced tight junction permeability in HUVECs monolayer which was attenuated by dLGG treatment. Furthermore, VEGF-induced melanoma cell extravasation via activation of FAK/PI3K/AKT pathway could be also inhibited by dLGG treatment. Oxylipins, a specific group of lipid mediators, play a crucial role in inflammation and are also implied in cancer and other diseases. We have established oxylipin metabolomics platform using UPLC/triple-quadrupole mass spectrometry coupled with partial least squares discriminant analysis (PLS-DA), to investigate the role of oxylipins involved in melanoma lung metastasis mice. The results of serum oxylipins analysis showed that cisplatin treatment not only induced significantly on anti-inflammatory 11,12-EET, but also increased proinflammatory 11-HETE and LTB4 (10- to 20-fold), relative to the tumor control. dLGG alone or dLGG and cisplatin combination treatment increased the anti-inflammatory EPA, EETs, 15-HEPE and 17-HDOHE (2- to 13-fold) in mouse serum. This study sheds light on the novel activity and the underlying molecular mechanisms of phytoagent dLGG on inhibition of melanoma cell EMT, tight junction permeability, and extravasation that support in part the in vivo therapeutic effect of dLGG on melanoma lung metastasis. Citation Format: Chung-Chih Yang, Meng-Ting Chang, Lie-Fen Shyur. Plant galactolipid dLGG suppresses lung metastasis of melanoma through modulating endothelial-mesenchymal transition extravasation and oxylipins dynamics. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A111.

Molecular Design of Bisphosphonate-Modified Proteins for Efficient Bone Targeting In Vivo.

To establish a rational molecular design for bisphosphonate (BP)-modified proteins for efficient bone targeting, a pharmacokinetic study was performed using a series of alendronate (ALN), a nitrogen-containing BP, modified proteins with various molecular weights and varying degrees of modification. Four proteins with different molecular weight—yeast glutathione reductase (GR; MW: 112,000 Da), bovine serum albumin (BSA; MW: 67,000 Da), recombinant human superoxide dismutase (SOD; MW: 32,000 Da), and chicken egg white lysozyme (LZM; MW: 14,000 Da)—were modified with ALN to obtain ALN-modified proteins. Pharmacokinetic analysis of the tissue distribution of the ALN-modified and unmodified proteins was performed after radiolabeling them with indium-111 (111In) by using a bifunctional chelating agent. Calculation of tissue uptake clearances revealed that the bone uptake clearances of 111In-ALN-modified proteins were proportional to the degree of ALN modification. 111In-GR-ALN and BSA-ALN, the two high-molecular-weight proteins, efficiently accumulated in bones, regardless of the degree of ALN modification. Approximately 36 and 34% of the dose, respectively, was calculated to be delivered to the bones. In contrast, the maximum amounts taken up by bone were 18 and 13% of the dose for 111In-SOD-ALN(32) and LZM-ALN(9), respectively, because of their high renal clearance. 111In-SOD modified with both polyethylene glycol (PEG) and ALN (111In-PEG-SOD-ALN) was efficiently delivered to the bone. Approximately 36% of the dose was estimated to be delivered to the bones. In an experimental bone metastasis mouse model, treatment with PEG-SOD-ALN significantly reduced the number of tumor cells in the bone of the mice. These results indicate that the combination of PEG and ALN modification is a promising approach for efficient bone targeting of proteins with a high total-body clearance.

Abstract 4113: Characterization of a novel TIMP-2 deficient mouse model of experimental lung metastasis

Abstract It is well known that the balance between matrix metalloproteinases (MMPs) and their endogenous inhibitors, the tissue inhibitors of metalloproteases (TIMPs) impacts tumor progression, thereby providing a novel avenue for cancer therapies. We have previously shown that forced TIMP-2 expression inhibits lung tumor growth independent of MMP inhibition via direct effects on tumor cells and modulation of the tumor microenvironment. The current study was undertaken to examine how reduced TIMP-2 expression impacts lung tumor growth using a novel TIMP-2 deficient (T2D) mouse model in a clean C57BL6 background. This is first time we fully developed and characterized TIMP2 deficient mice, validated by genotyping, histologic staining, protein and mRNA expression. The deletion of TIMP2 gene down regulated the expression TIMP2 mRNA and protein as demonstrated by quantitative RT-PCR and immunoblot experiments, respectively. Further, we investigated the impact of TIMP2 deletion on Lewis lung carcinoma model of metastasis. Lewis lung carcinoma cells transfected with luciferase (LL/2-Luc-M38, Caliper) were intratracheally instilled in the lungs into group's have heterozygous (T2D+/−), homozygous (T2D−/−), and wild type (WT) littermates. Tumor progression was monitored by IVIS imaging after 2nd week of intratracheal instillation and continued till 4th week, mice were sacrificed and lungs were perfused and harvested for further analysis. The IVIS analysis showed higher tumor burden in TIMP2 deficient and heterozygous mice as compared to wild type littermate, suggested that TIMP2 deficiency aggravates tumor development in deficient mice. The effects of recombinant TIMP2 on tumor microenvironment and microarray experiments are currently in progress. The molecular mechanism underlying the observed activities will be investigated. Collectively, these preliminary findings suggest that TIMP2 may be a novel target for lung cancer therapy. Citation Format: Sarvesh Kumar. Characterization of a novel TIMP-2 deficient mouse model of experimental lung metastasis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4113. doi:10.1158/1538-7445.AM2015-4113

Cadherin-11 regulates the metastasis of Ewing sarcoma cells to bone.

Ewing sarcoma (ES) is a small round-cell tumor of the bones and soft tissues. ES frequently causes distant metastases, particularly in the lung and bone, which worsens patient prognosis. Cadherin-11 (Cad-11) is an adhesion molecule that is highly expressed in osteoblasts. Its expression is associated with bone metastases in prostate and breast cancer patients, and is known to occur in ES. Here we investigated the effects of Cad-11 on bone metastases of ES. Human ES cell lines RD-ES, SK-ES-1, SK-N-MC, and TC-71 cells were transduced with lentivirus containing Cad-11 shRNA or control shRNA (ES/Cad-11 and ES/Ctr). RD-ES and TC-71 were infected with a lentivirus luciferase vector. Adhesion assays were performed using these cells and recombinant Cad-11-Fc chimera or mouse osteoblast cell line MC3T3-E1. Cell motility was investigated via wound-healing assay. Intracardiac injection of RD-ES/Cad-11 and RD-ES/Ctr was used to create a mouse model of experimental bone metastasis. The association between Cad-11 expression and bone metastases and clinical prognosis in ES patients was analyzed by immunohistochemistry. We found knockdown of Cad-11 in ES cells resulted in reduced attachment ability and cell motility. In a mouse model of metastasis, RD-ES/Cad-11 cells caused fewer metastases than RD-ES/Ctr cells. The expression of Cad-11 in ES patients was significantly related to bone metastases (P < 0.05, logistic regression) and poorer overall survival (P < 0.05, log-rank test). These findings may explain that Cad-11 in ES cells may be essential for cell adhesion and motility, and is a promising molecular target for patients with ES.

Inhibition of spontaneous and experimental lung metastasis of soft-tissue sarcoma by tumor-targeting Salmonella typhimurium A1-R.

Prognosis of patients with lung metastases of soft-tissue sarcoma is still poor. Therefore, novel systemic therapy is needed to improve the survival of soft-tissue sarcoma. In the present study, tumor-targeting therapy with a genetically-modified auxotrophic strain of Salmonella typhimurium, termed A1-R, was evaluated. Mouse models of primary soft tissue sarcoma and spontaneous lung metastasis were obtained by orthotopic intra-muscular injection of HT1080-RFP human fibrosarcoma cells. S. typhimurium A1-R was administered from day 14, once a week for two weeks. On day 28, lung samples were excised and observed with a fluorescence imaging system. The number of lung metastasis was 8.8 ± 3.4 in the untreated group and 0.8 ± 0.8 in the treated group (P = 0.024). A mouse model of experimental lung metastasis was obtained by tail vein injection of HT1080-RFP cells. The mice were treated with S. typhimurium A1-R (i.v.) on day 7, once a week for three weeks. S. typhimurium A1-R significantly reduced lung metastases and improved overall survival (P = 0.004). S. typhimurium A1-R bacterial therapy has future potential for treating advanced soft tissue sarcoma and improving prognosis of patients with lung metastasis.

The Rac Inhibitor EHop-016 Inhibits Mammary Tumor Growth and Metastasis in a Nude Mouse Model

Metastatic disease still lacks effective treatments, and remains the primary cause of cancer mortality. Therefore, there is a critical need to develop better strategies to inhibit metastatic cancer. The Rho family GTPase Rac is an ideal target for anti-metastatic cancer therapy, because Rac is a key molecular switch that is activated by a myriad of cell surface receptors to promote cancer cell migration/invasion and survival. Previously, we reported the design and development of EHop-016, a small molecule compound, which inhibits Rac activity of metastatic cancer cells with an IC50 of 1μM. EHop-016 also inhibits the activity of the Rac downstream effector p21-activated kinase (PAK), lamellipodia extension, and cell migration in metastatic cancer cells. Herein, we tested the efficacy of EHop-016 in a nude mouse model of experimental metastasis, where EHop-016 administration at 25mg/kg body weight (BW) significantly reduced mammary fat pad tumor growth, metastasis, and angiogenesis. As quantified by UPLC MS/MS, EHop-016 was detectable in the plasma of nude mice at 17 to 23ng/ml levels at 12h following intraperitoneal (i.p.) administration of 10 to 25mg/kg BW EHop-016. The EHop-016 mediated inhibition of angiogenesis In Vivo was confirmed by immunohistochemistry of excised tumors and by In Vitro tube formation assays of endothelial cells. Moreover, EHop-016 affected cell viability by down-regulating Akt and Jun kinase activities and c-Myc and Cyclin D expression, as well as increasing caspase 3/7 activities in metastatic cancer cells. In conclusion, EHop-016 has potential as an anticancer compound to block cancer progression via multiple Rac-directed mechanisms.

Abstract 2756: Ceramide analog targets xIAP and cIAP1 to sensitize metastatic colon carcinoma cells to apoptosis induction to suppress tumor progression

Abstract Sphingolipids are bioeffectors that mediate various cellular processes. We aimed to identify molecular targets of ceramide in the apoptosis pathway and to determine the efficacy of a ceramide analog in overcoming cancer cell resistance to apoptosis induction. We observed that exposure of human colon carcinoma cells to ceramide analog LCL85 results in apoptosis in a dose-dependent manner. Interestingly, a sublethal dose of LCL85 increased C16 ceramide content and overcame tumor cell resistance to FasL-induced apoptosis. Subsequently, treatment of tumor cells with exogenous C16 ceramide resulted in increased tumor cell sensitivity to FasL-induced apoptosis. LCL85 resembles Smac mimetic in sensitization of colon carcinoma cells to FasL-induced apoptosis by inducing proteasomal degradation of cIAP1 and xIAP proteins. Consistent with its apoptosis sensitization activity, LCL85 suppressed colon carcinoma cell metastatic potential in an experimental colon carcinoma lung metastasis mouse model. Taken together, we determined that a sublethal dose of LCL85 is effective as a sensitizer in overcoming apoptosis resistance of metastatic human colon carcinoma cells, and LCL85 is effective in suppressing tumor metastasis in vivo. Citation Format: Amy Paschall, Mary Zimmerman, Christina Torres, Dafeng Yang, May Chen, Xia Li, Erhard Bieberich, Aiping Bai, Jacek Bielawski, Alicja Bielawska, Kebin Liu. Ceramide analog targets xIAP and cIAP1 to sensitize metastatic colon carcinoma cells to apoptosis induction to suppress tumor progression. [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 2756. doi:10.1158/1538-7445.AM2014-2756

Vasodilator-stimulated phosphoprotein promotes activation of hepatic stellate cells by regulating Rab11-dependent plasma membrane targeting of transforming growth factor beta receptors.

Liver microenvironment is a critical determinant for development and progression of liver metastasis. Under transforming growth factor beta (TGF-β) stimulation, hepatic stellate cells (HSCs), which are liver-specific pericytes, transdifferentiate into tumor-associated myofibroblasts that promote tumor implantation (TI) and growth in the liver. However, the regulation of this HSC activation process remains poorly understood. In this study, we tested whether vasodilator-stimulated phosphoprotein (VASP) of HSCs regulated the TGF-β-mediated HSC activation process and tumor growth. In both an experimental liver metastasis mouse model and cancer patients, colorectal cancer cells reaching liver sinusoids induced up-regulation of VASP and alpha-smooth muscle actin (α-SMA) in adjacent HSCs. VASP knockdown in HSCs inhibited TGF-β-mediated myofibroblastic activation of HSCs, TI, and growth in mice. Mechanistically, VASP formed protein complexes with TGF-β receptor II (TβRII) and Rab11, a Ras-like small GTPase and key regulator of recycling endosomes. VASP knockdown impaired Rab11 activity and Rab11-dependent targeting of TβRII to the plasma membrane, thereby desensitizing HSCs to TGF-β1 stimulation. Our study demonstrates a requirement of VASP for TGF-β-mediated HSC activation in the tumor microenvironment by regulating Rab11-dependent recycling of TβRII to the plasma membrane. VASP and its effector, Rab11, in the tumor microenvironment thus present therapeutic targets for reducing TI and metastatic growth in the liver.

Cabozantinib Suppresses Tumor Growth and Metastasis in Hepatocellular Carcinoma by a Dual Blockade of VEGFR2 and MET

MET signaling has been suggested a potential role in hepatocellular carcinoma (HCC) and associated with prometastasis during antiangiogenesis therapy. We investigated the potential association between MET expression and therapeutic response to sorafenib in patients with HCC. Antitumor effects of cabozantinib, a dual inhibitor of MET and VEGFR2, were examined in cultured HCC cells as well as in vivo models. Total MET and phosphorylated MET (p-MET) were measured in 29 resected HCC specimens, and correlated with response to sorafenib as postoperative adjuvant therapy. In the second set of experiments using cultured HCC cells, and mouse xenograft and metastatic models, effects of cabozantinib were examined. High level of p-MET in resected HCC specimens was associated with resistance to adjuvant sorafenib therapy. In cultured HCC cells that expressed p-MET, cabozantinib inhibited the activity of MET and its downstream effectors, leading to G1-phase arrest. Cabozantinib inhibited tumor growth in p-MET-positive and p-MET-negative HCC by decreasing angiogenesis, inhibiting proliferation, and promoting apoptosis, but it exhibited more profound efficacy in p-MET-positive HCC xenografts. Cabozantinib blocked the hepatocyte growth factor (HGF)-stimulated MET pathway and inhibited the migration and invasion of the HCC cells. Notably, cabozantinib reduced the number of metastatic lesions in the lung and liver in the experimental metastatic mouse model. Patients with HCC with high level of p-MET are associated with resistance to adjuvant sorafenib treatment. The dual blockade of VEGFR2 and MET by cabozantinib has significant antitumor activities in HCC, and the activation of MET in HCC may be a promising efficacy-predicting biomarker. Clin Cancer Res; 20(11); 2959-70. ©2014 AACR.

Determination of Rac inhibitor Ehop‐016 in mouse plasma by ultra‐performance liquid chromatography tandem mass spectrometry (987.2)

The Rho GTPase Rac is an important regulator of cancer cell migration and cell invasion; processes required for metastatic progression. We previously characterized the small molecule Ehop‐016 as a novel Rac inhibitor in metastatic breast cancer cells in‐vitro (Montalvo‐Ortiz, et al., 2012). To investigate the efficacy of EHop‐016 in‐vivo, we used a mouse model of experimental metastasis, where mice with MDA‐MB‐435 mammary fat pad tumors were treated with intraperitoneal EHop‐016. At 25 and 40 mg/kg BW, EHop‐016 significantly inhibited mammary tumor growth and metastasis. In order to characterize the pharmacokinetics of Ehop‐016, we developed a rapid and sensitive method for the quantitation of Ehop‐016 in mouse plasma by ultra high performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry (UPLC/MS/MS). Plasma samples were pretreated with acetonitrile (ACN) to extract matrix proteins prior to injection into UPLC/MS/MS. Separation was carried out on an Agilent Poroshell 120 EC‐C18 column (3.0 x 50) mm using a mobile phase of 50% ACN/50% methanol/0.1% formic acid and 1mM ammonium fluoride. Ehop‐016 was identified from its accurate mass and retention times from the acquired full‐scan chromatogram and quantified by its peak areas. The linear range for the determination of analytes was 5 ‐ 1000 ng/mL. Ehop‐016 was quantified in samples from the in‐vivo study using this method.

Delivery of Therapeutic siRNA to the Lung Endothelium via Novel Lipoplex Formulation DACC

Posttranscriptional gene silencing by RNA interference can be therapeutically exploited to inhibit pathophysiological gene expression. However, in contrast to the established effectiveness of RNAi in vitro, safe and effective delivery of siRNAs to specific organs and cell types in vivo remains the major hurdle. Here, we report the development and in vivo characterization of a novel siRNA delivery system (DACC lipoplex) suitable for modulating target gene expression specifically in the lung vasculature. Systemic administration of DACC in mice delivered siRNA cargo functionally to the lung pulmonary endothelium. A single dose of DACC lipoplexes administered by bolus injection or by infusion was sufficient to specifically silence genes expressed in pulmonary endothelial cells such as CD31, Tie-2, VE-cadherin, or BMP-R2. When tested in a mouse model for lung cancer, repeated treatment with DACC/siRNA(CD31) reduced formation of lung metastases and increased life span in a mouse model of experimental lung metastasis.

Ceramide targets xIAP and cIAP1 to sensitize metastatic colon and breast cancer cells to apoptosis induction to suppress tumor progression

BackgroundCeramide is a bioeffector that mediates various cellular processes, including apoptosis. However, the mechanism underlying ceramide function in apoptosis is apparently cell type-dependent and is not well-understood. We aimed at identifying molecular targets of ceramide in metastatic human colon and breast cancer cells, and determining the efficacy of ceramide analog in suppression of colon and breast cancer metastasis.MethodsThe activity of and mechanism underlying ceramide as a cytotoxic agent, and as a sensitizer for Fas-mediated apoptosis was analyzed in human cell lines established from primary or metastatic colon and breast cancers. The efficacy of ceramide analog LCL85 in suppression of metastasis was examined in preclinical mouse tumor models.ResultsExposure of human colon carcinoma cells to ceramide analog LCL85 results in apoptosis in a dose-dependent manner. Interestingly, a sublethal dose of LCL85 increased C16 ceramide content and overcame tumor cell resistance to Fas-mediated apoptosis. Subsequently, treatment of tumor cells with exogenous C16 ceramide resulted in increased tumor cell sensitivity to Fas-mediated apoptosis. LCL85 resembles Smac mimetic BV6 in sensitization of colon carcinoma cells to Fas-mediated apoptosis by inducing proteasomal degradation of cIAP1 and xIAP proteins. LCL85 also decreased xIAP1 and cIAP1 protein levels and sensitized metastatic human breast cancer cells to Fas-mediated apoptosis. Silencing xIAP and cIAP1 with specific siRNAs significantly increased the metastatic human colon carcinoma cell sensitivity to Fas-mediated apoptosis, suggesting that IAP proteins mediate apoptosis resistance in metastatic human colon carcinoma cells and ceramide induces IAP protein degradation to sensitize the tumor cells to apoptosis induction. Consistent with its apoptosis sensitization activity, subtoxic doses of LCL85 suppressed colon carcinoma cell metastatic potential in an experimental lung metastasis mouse model, as well as breast cancer growth and spontaneous lung metastasis in an orthotopic breast cancer mouse model.ConclusionWe have identified xIAP and cIAP1 as molecular targets of ceramide and determined that ceramide analog LCL85 is an effective sensitizer in overcoming resistance of human cell lines established from metastatic colon and breast cancers to apoptosis induction to suppress metastasis in vivo.

Contributions of thrombin targets to tissue factor‐dependent metastasis in hyperthrombotic mice

Tumor cell tissue factor (TF)-initiated coagulation supports hematogenous metastasis by fibrin formation, platelet activation and monocyte/macrophage recruitment. Recent studies identified host anticoagulant mechanisms as a major impediment to successful hematogenous tumor cell metastasis. Here we address mechanisms that contribute to enhanced metastasis in hyperthrombotic mice with functional thrombomodulin deficiency (TM(Pro) mice). Pharmacological and genetic approaches were combined to characterize relevant thrombin targets in a mouse model of experimental hematogenous metastasis. TF-dependent, but contact pathway-independent, syngeneic breast cancer metastasis was associated with marked platelet hyperreactivity and formation of leukocyte-platelet aggregates in immune-competent TM(Pro) mice. Blockade of CD11b or genetic deletion of platelet glycoprotein Ibα excluded contributions of these receptors to enhanced platelet-dependent metastasis in hyperthrombotic mice. Mice with very low levels of the endothelial protein C receptor (EPCR) did not phenocopy the enhanced metastasis seen in TM(Pro) mice. Genetic deletion of the thrombin receptor PAR1 or endothelial thrombin signaling targets alone did not diminish enhanced metastasis in TM(Pro) mice. Combined deficiency of PAR1 on tumor cells and the host reduced metastasis in TM(Pro) mice. Metastasis in the hyperthrombotic TM(Pro) mouse model is mediated by platelet hyperreactivity and contributions of PAR1 signaling on tumor and host cells.

Abstract A155: The Rac and PAK inhibitor EHop-016 inhibits mammary tumor growth and metastasis in a nude mouse model.

Abstract The Rho GTPase Rac is a central regulator of cancer cell migration/invasion that has been implicated with cancer metastasis. Therefore, we designed and developed small molecule compounds that specifically target Rac in metastatic cancer cells. We recently reported the characterization of EHop-016 as a Rac inhibitor in metastatic cancer cells, where EHop-016 was shown to inhibit Rac activity with an IC50 of 1 μM. At higher concentrations (&amp;gt;10 μM) EHop-016 reduced cell viability and inhibited the related Rho GTPase Cdc42, but not Rho. EHop-016 decreased the activation of Rac by the oncogenic guanine nucleotide exchange factor Vav, the activity of the Rac effector p21-activated kinase (PAK), and the extension of actin cytoskeletal structures and migration of metastatic cancer cells. Next, we determined the efficacy of EHop-016 in vivo, using a mouse model of experimental metastasis. Green fluorescent protein (GFP) tagged MDA-MB-435 human metastatic cancer cells were inoculated at the mammary fat pad of nude mice and treated with 0, 5, 10, 25, or 40 mg/kg body weight (BW) EHop-016 3X a week for ∼8 weeks. Mammary tumor growth and metastases in lungs, spleens, kidneys, livers, hearts, and femurs were quantified by fluorescence image analysis. At concentrations &amp;gt;10 mg/kg BW, EHop-016 significantly inhibited mammary tumor growth and metastasis to distant organs. EHop-016 had no effect on mouse weight or their gross phenotype, indicating that EHop-016 did not exert toxic effects in athymic nude mice. This data demonstrate that EHop-016 and derivatives hold promise for further development as targeted anti-cancer therapeutics. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A155. Citation Format: Linette Castillo-Pichardo, Tessa Humphries-Bickley, Eliud Hernandez, Columba de la Parra, Luis Cubano, Cornelis Vlaar, Surangani F. Dharmawardhane Flanagan. The Rac and PAK inhibitor EHop-016 inhibits mammary tumor growth and metastasis in a nude mouse model. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A155.

Suberoylanilide Hydroxamic Acid, an Inhibitor of Histone Deacetylase, Enhances Radiosensitivity and Suppresses Lung Metastasis in Breast Cancer In Vitro and In Vivo

Triple-negative breast cancer (TNBC), defined by the absence of an estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 expression, is associated with an early recurrence of disease and poor outcome. Furthermore, the majority of deaths in breast cancer patients are from metastases instead of from primary tumors. In this study, MCF-7 (an estrogen receptor-positive human breast cancer cell line), MDA-MB-231 (a human TNBC cell line) and 4T1 (a mouse TNBC cell line) were used to investigate the anti-cancer effects of ionizing radiation (IR) combined with suberoylanilide hydroxamic acid (SAHA, an inhibitor of histone deacetylase (HDAC)) and to determine the underlying mechanisms of these effects in vitro and in vivo. We also evaluated the ability of SAHA to inhibit the metastasis of 4T1 cells. We found that IR combined with SAHA showed increased therapeutic efficacy when compared with either treatment alone in MCF-7, MDA-MB-231 and 4T1 cells. Moreover, the combined treatment enhanced DNA damage through the inhibition of DNA repair proteins. The combined treatment was induced primarily through autophagy and ER stress. In an orthotopic breast cancer mouse model, the combination treatment showed a greater inhibition of tumor growth. In addition, SAHA inhibited the migration and invasion abilities of 4T1 cells and inhibited breast cancer cell migration by inhibiting the activity of MMP-9. In an in vivo experimental metastasis mouse model, SAHA significantly inhibited lung metastasis. SAHA not only enhances radiosensitivity but also suppresses lung metastasis in breast cancer. These novel findings suggest that SAHA alone or combined with IR could serve as a potential therapeutic strategy for breast cancer.

FK506 binding protein 51 positively regulates melanoma stemness and metastatic potential

Melanoma is the most aggressive skin cancer; there is no cure in advanced stages. Identifying molecular participants in melanoma progression may provide useful diagnostic and therapeutic tools. FK506 binding protein 51 (FKBP51), an immunophilin with a relevant role in developmental stages, is highly expressed in melanoma and correlates with aggressiveness and therapy resistance. We hypothesized a role for FKBP51 in melanoma invasive behaviour. FKBP51 promoted activation of epithelial-to-mesenchymal transition (EMT) genes and improved melanoma cell migration and invasion. In addition, FKBP51 induced some melanoma stem cell (MCSC) genes. Purified MCSCs expressed high EMT genes levels, suggesting that genetic programs of EMT and MCSCs overlap. Immunohistochemistry of samples from patients showed intense FKBP51 nuclear signal and cytoplasmic positivity for the stem cell marker nestin in extravasating melanoma cells and metastatic brains. In addition, FKBP51 targeting by small interfering RNA (siRNA) prevented the massive metastatic substitution of liver and lung in a mouse model of experimental metastasis. The present study provides evidence that the genetic programs of cancer stemness and invasiveness overlap in melanoma, and that FKBP51 plays a pivotal role in sustaining such a program.

Lymphotoxin β receptor mediates caspase-dependent tumor cell apoptosis in vitro and tumor suppression in vivo despite induction of NF-κB activation

Ligation of the lymphotoxin β receptor (LTβR) has been shown to induce both tumor growth inhibition and promotion. The functions of LTβR in these two contrasting cellular processes require further study. We demonstrated here that mice deficient in LTβ R ligands, LTα, LIGHT or both LTβ and LIGHT, exhibit greater susceptibility to methylcholanthrene-induced tumor development. LTα, LTβ and LIGHT were expressed in tumor-infiltrating immune cells, and LTβR was expressed on human colon carcinoma and soft tissue sarcoma (STS) cells. Human LTβR agonist monoclonal antibody (mAb) BS-1 induced both growth inhibition and NF-κB activation in human colon carcinoma, mammary carcinoma and STS cells. Interestingly, BS-1 also significantly inhibited growth of doxorubicin-resistant and radiation-resistant human STS cells in vitro. In the molecular mechanism level, we demonstrated that BS-1 induces caspases 8 and 3 activation and cytochrome c release in tumor cells, suggesting that the LTβR mediates apoptosis at least partially through a caspase-dependent mechanism. Furthermore, mouse LTβR mAb ACH6 suppressed colon carcinoma cell metastatic potential in an experimental metastasis mouse model. Although blocking NF-κB activation did not alter tumor cell growth rate and tumor cell response to LTβR mAb-induced growth inhibition in vitro, surprisingly, blocking NF-κB activation significantly enhanced colon carcinoma cell metastatic potential in vivo, suggesting that the LTβR-mediated apoptosis pathway and NF-κB signaling pathway might cooperate to suppress tumor growth in vivo. In summary, our findings deter mine that LTβR mediates tumor cell apoptosis in colon carcinoma, mammary carcinoma and sarcoma and that LTβR-activated NF-κB potentially functions as a tumor suppressor.

Effect of Inhibition of the Lysophosphatidic Acid Receptor 1 on Metastasis and Metastatic Dormancy in Breast Cancer

Previous studies identified the human nonmetastatic gene 23 (NME1, hereafter Nm23-H1) as the first metastasis suppressor gene. An inverse relationship between Nm23-H1 and expression of lysophosphatidic acid receptor 1 gene (LPAR1, also known as EDG2 or hereafter LPA1) has also been reported. However, the effects of LPA1 inhibition on primary tumor size, metastasis, and metastatic dormancy have not been investigated. The LPA1 inhibitor Debio-0719 or LPA1 short hairpinned RNA (shRNA) was used. Primary tumor size and metastasis were investigated using the 4T1 spontaneous metastasis mouse model and the MDA-MB-231T experimental metastasis mouse model (n = 13 mice per group). Proliferation and p38 intracellular signaling in tumors and cell lines were determined by immunohistochemistry and western blot to investigate the effects of LPA1 inhibition on metastatic dormancy. An analysis of variance-based two-tailed t test was used to determine a statistically significant difference between treatment groups. In the 4T1 spontaneous metastasis mouse model, Debio-0719 inhibited the metastasis of 4T1 cells to the liver (mean = 25.2 liver metastases per histologic section for vehicle-treated mice vs 6.8 for Debio-0719-treated mice, 73.0% reduction, P < .001) and lungs (mean = 6.37 lesions per histologic section for vehicle-treated mice vs 0.73 for Debio-0719-treated mice, 88.5% reduction, P < .001), with no effect on primary tumor size. Similar results were observed using the MDA-MB-231T experimental pulmonary metastasis mouse model. LPA1 shRNA also inhibited metastasis but did not affect primary tumor size. In 4T1 metastases, but not primary tumors, expression of the proliferative markers Ki67 and pErk was reduced by Debio-0719, and phosphorylation of the p38 stress kinase was increased, indicative of metastatic dormancy. The data identify Debio-0719 as a drug candidate with metastasis suppressor activity, inducing dormancy at secondary tumor sites.

Regulation of motility, invasion, and metastatic potential of squamous cell carcinoma by 1α,25‐dihydroxycholecalciferol

The active metabolite of vitamin D 1α,25-dihydroxycholecalciferol (1,25D(3) ) has exhibited broad-spectrum antitumor activity in xenograft animal models. However, its activity against metastatic disease has not been extensively investigated. Squamous cell carcinoma (SCC) or 1,25D(3) -resistant variant SCC-DR cells were treated with 1,25D(3) . Actin organization was examined by immunofluorescence assay. Cell migration was assessed by "wound" healing and chemotactic migration assays. Cell invasion was assessed by a Matrigel-based invasion assay and in situ zymography. Matrix metalloproteinase 2 (MMP-2) and MMP-9 expression and secretion were examined by immunoblot analysis and an enzyme-linked immunosorbent assay, respectively. E-cadherin expression was assessed by flow cytometry, immunoblot analysis, and immunohistochemistry. Knockdown of E-cadherin was achieved by small interfering RNA. An experimental metastasis mouse model was created by intravenous injection of tumor cells; and lung tumor development in the mice was assessed by magnetic resonance imaging, gross observation, and histology. SCC cellular morphology and actin organization were altered by 10 nM 1,25D(3) . 1,25D(3) inhibited SCC cell motility and invasion, which were associated with reduced expression and secretion of MMP-2 and MMP-9, and 1,25D(3) promoted the expression of E-cadherin. These findings were not observed in SCC-DR cells. Knock down of E-cadherin rescued 1,25D(3) -inhibited cell migration. Intravenous injection of SCC or SCC-DR cells resulted in the establishment of extensive pulmonary lesions in saline-treated C3H mice. Treatment with 1,25D(3) resulted in a marked reduction in the formation of lung tumor colonies in mice that were injected with SCC cells, but not in mice that were injected with SCC-DR cells. 1,25D(3) suppressed SCC cell motility, invasion, and metastasis, partially through the promotion of E-cadherin-mediated cell-cell adhesion.

The dominant expression of functional human lactoferrin in transgenic cloned goats using a hybrid lactoferrin expression construct

Human Lactoferrin (hLF) is an iron-binding protein with multiple physiological functions. As the availability of natural hLF is limited, alternative means of producing this biopharmaceutical protein have been extensively studied. Here we report on the dominant expression of recombinant human lactoferrin (rhLF) in transgenic cloned goats using a novel optimised construct made by fusing a 3.3kb hLF minigene to the regulatory elements of the β-casein gene. The transgenic goat produced more than 30mg/ml rhLF in its milk, and rhLF expression was stable during the entire lactation cycle. The rhLF purification efficiency from whole goat milk is approximately 70%, and its purity is above 98%. Compared with natural hLF, the rhLF from transgenic goats has similar biological characteristics including molecular mass, N-terminal sequence, isoelectric point, immunoreactivity and digestive stability. More importantly, the purified rhLF showed specific anti-tumour activity in the mouse model of melanoma experimental metastasis. Therefore, our study shows that the large-scale production of functional rhLF in transgenic goat milk could be an economical and promising source of human therapeutic use in the future.