Murine Mammary Carcinoma Research Articles

WE‐E‐BRE‐06: High‐Dose Microbeam Radiation Induces Different Responses in Tumor Microenvironment Compared to Conventional Seamless Radiation in Window Chamber Tumor Models

Purpose:Microbeam radiation therapy and GRID therapy are different forms of Spatially‐Fractioned Radiation Therapy (SFRT) that is fundamentally different from the conventional seamless and temporally fractionated radiation therapy. SFRT is characterized by a ultra‐high dose (10s –100s Gy) dose single treatment with drastic inhomogeneity pattern of given spatial frequencies. Preclinical and limited clinical studies have shown that the SFRT treatments may offer significant improvements in reducing treatment toxicity, especially for those patients who have not benefited from the state‐of‐the‐art radiation therapy approaches. This preliminary study aims to elucidate the underlying working mechanisms of SFRT, which currently remains poorly understood.Methods:A genetically engineered 4T1 murine mammary carcinoma cell line and nude mice skin fold window chamber were used. A nanotechnology‐based 160kV x‐ray irradiator delivered 50Gy (entrance dose) single treatments of microbeam or seamless radiation. Animals were in 3 groups: mock, seamless radiation, and 300μm microbeam radiation. The windows were imaged using a hyperspectral system to capture total hemoglobin/saturation, GFP fluorescence emission, RFP fluorescence emission, and vessel density at 9 time points up to 7 days post radiation.Results:We found unique physiologic changes in different tumor/normal tissue regions and differential effects between seamless and microbeam treatments. They include 1) compared to microbeam and mock radiation seamless radiation damaged more microvasculature in tumor‐surrounding normal tissue, 2) a pronounced angiogenic effect was observed with vascular proliferation in the microbeam irradiated portion of the tumor days post treatment (no such effect observed in seamless and mock groups), and 3) a notable change in tumor vascular orientation was observed where vessels initially oriented parallel to the beam length were replaced by vessels running perpendicular to the irradiation portion of the tumor.Conclusion:Our preliminary study indicated that microbeam radiation modified tumor microenvironment in ways significantly different than of the conventional seamless radiation.

Improving the distribution of Doxil® in the tumor matrix by depletion of tumor hyaluronan

Liposomes improve the pharmacokinetics and safety of rapidly cleared drugs, but have not yet improved the clinical efficacy compared to the non-encapsulated drug. This inability to improve efficacy may be partially due to the non-uniform distribution of liposomes in solid tumors. The tumor extra-cellular matrix is a barrier to distribution and includes the high molecular weight glycosaminoglycan, hyaluronan (HA). Strategies to remove HA or block its synthesis may improve drug delivery into solid tumors. Orally administered methylumbelliferone (MU) is an inhibitor of HA synthesis, but it is limited by low potency and limited solubility. In this study, we encapsulate a water-soluble phosphorylated prodrug of MU (MU-P) in a liposome (L-MU-P). We demonstrate that L-MU-P is a more potent inhibitor of HA synthesis than oral MU in the 4T1 murine mammary carcinoma model using both a quantitative ELISA and histochemistry. We show that HA depletion improves the tumor distribution of liposomes computed using Mander's colocalization analysis of liposomes with the tumor vasculature. Hyaluronan depletion also increases the fraction of the tumor area positive for liposomes. This improved distribution extends the overall survival of mice treated with Doxil®.

Constitutive signaling through Myd88 contributes to tumor-derived CCL2 expression and nuclear translocation of NFkB p65, p52, Fos and ATF2 (TUM7P.923)

Abstract Previous data in our lab suggested that there may be constitutive signaling through the Myd88 signaling cascade in murine mammary carcinoma. Here we extend these findings by showing that the myddosome complex is preformed in the tumor cells. Using co-immunoprecipitation we found Myd88 associated with IRAK2 and IRAK4. After screening many different products of the TLR signaling cascade we found that the most clear consequence of this Myd88 signaling in the tumor cells was expression of CCL2. Targeting Myd88 expression using RNA interference, or function using an inhibitory peptide, resulted in reduced CCL2 expression in a dose dependent manner. Coinciding with Myd88 dependent expression of CCL2 we also detected presence of the CCL2 transcriptional factors NFkB p65, p52, Fos and ATF2 in the nucleus of the tumor cells, and the levels of these proteins in the nucleus decreased upon treatment with the Myd88 inhibitory peptide. Targeting Myd88 using the inhibitory peptide, constitutive shRNA expression, or tetracycline inducible shRNA expression also resulted in a reduced tumor growth rate in vitro. Despite this we found limited therapeutic potential of targeting Myd88 in vivo using the inhibitory peptide alone. Collectively, these data imply that constitutive Myd88 signaling contributes to tumor-derived CCL2 expression, and suggests that alternative mechanisms to target Myd88 in vivo warrants investigation.

PEGylation alleviates the non-specific toxicities of Alpha-Momorcharin and preserves its antitumor efficacy in vivo

Alpha-Momorcharin (α-MMC) is a ribosome inactivating protein from Momordica charantia with anti-tumor activity. Previously, we had observed that modification of α-MMC with polyethylene glycol (PEG) could reduce toxicity, but it also reduces its anti-tumor activity in vitro. This study aims to investigate whether the metabolism-extended properties of α-MMC resulting from PEGylation could preserve its anti-tumor efficacy in vivo through pharmacokinetics and antitumor experiments. The pharmacokinetics experiments were conducted in rats using the TCA (Trichloroacetic Acid) method. Antitumor activity in vivo was investigated in murine mammary carcinoma (EMT-6) and human mammary carcinoma (MDA-MB-231) transplanted tumor mouse models. The results showed that PEGylation increased the plasma half-life of α-MMC in rats from 6.2–7.5 h to 52–87 h. When administered at 1 mg/kg, α-MMC-PEG and α-MMC showed similar anti-tumor activities in vivo, with a T/C% of 38.56% for α-MMC versus 35.43% for α-MMC-PEG in the EMT-6 tumor model and 36.30% for α-MMC versus 39.88% for α-MMC-PEG in the MDA-MB-231 tumor model (p > 0.05). Importantly, at the dose of 3 mg/kg, all the animals treated with α-MMC died while the animals treated with α-MMC-PEG exhibited only moderate toxic reactions, and α-MMC-PEG exhibited improved anti-tumor efficacy with a T/C% (relative tumor growth rate) of 25.18% and 21.07% in the EMT-6 and MDA-MB-231 tumor models, respectively. The present study demonstrates that PEGylation extends the half-life of α-MMC and alleviates non-specific toxicity, thereby preserving its antitumor efficacy in vivo, and a higher lever of dosage can be used to achieve better therapeutic efficacy.

The effectiveness of nano chemotherapeutic particles combined with mifepristone depends on the PR isoform ratio in preclinical models of breast cancer.

There is clinical and experimental evidence suggesting that antiprogestins might be used for the treatment of selected breast cancer patients. Our aim was to evaluate the effect of albumin-bound paclitaxel (Nab-paclitaxel) and pegylated doxorubicin liposomes (PEG-LD) in combination with mifepristone (MFP) in experimental breast cancer models expressing different ratios of progesterone receptor (PR) isoforms A and B. We used two antiprogestin-responsive (PRA>PRB) and two resistant (PRA<PRB) murine mammary carcinomas growing in BALB/c, GFP-BALB/c or nude mice, along with human T47D-YA and T47D-YB xenografts growing in immunocompromised NSG mice. MFP improved the therapeutic effects of suboptimal doses of Nab-paclitaxel or PEG-LD in murine and human carcinomas with higher levels of PRA than PRB. MFP induced tissue remodeling in PRA-overexpressing tumors, increasing the stromal/tumor cell ratio and the number of functional vessels. Accordingly, an increase in nanoparticles and drug accumulation was observed in stromal and tumor cells in MFP-treated tumors. We conclude that MFP induces an increase in vessels during tissue remodeling, favoring the selective accumulation of nanoparticles inside the tumors. We propose that antiprogestins have the potential to enhance the efficacy of chemotherapy in breast tumors with a high PRA/PRB ratio.

Cathepsin B: multiple roles in cancer.

Proteases, including intracellular proteases, play roles at many different stages of malignant progression. Our focus here is cathepsin B, a lysosomal cysteine cathepsin. High levels of cathepsin B are found in a wide variety of human cancers, levels that often induce secretion and association of cathepsin B with the tumor cell membrane. In experimental models, such as transgenic models of murine pancreatic and mammary carcinomas, causal roles for cathepsin B have been demonstrated in initiation, growth/tumor cell proliferation, angiogenesis, invasion, and metastasis. Tumor growth in transgenic models is promoted by cathepsin B in tumor-associated cells, for example, tumor-associated macrophages, as well as in tumor cells. In transgenic models, the absence of cathepsin B has been associated with enhanced apoptosis, yet cathepsin B also has been shown to contribute to apoptosis. Cathepsin B is part of a proteolytic pathway identified in xenograft models of human glioma; targeting only cathepsin B in these tumors is less effective than targeting cathepsin B in combination with other proteases or protease receptors. Understanding the mechanisms responsible for increased expression of cathepsin B in tumors and association of cathepsin B with tumor cell membranes is needed to determine whether targeting cathepsin B could be of therapeutic benefit.

Aluminum&amp;ndash;phthalocyanine chloride associated to poly(methyl vinyl ether-co-maleic anhydride) nanoparticles as a new third-generation photosensitizer for anticancer photodynamic therapy

Photodynamic therapy is generally considered to be safer than conventional anticancer therapies, and it is effective against different kinds of cancer. However, its clinical application has been significantly limited by the hydrophobicity of photosensitizers. In this work, a system composed of the hydrophobic photosensitizer aluminum–phthalocyanine chloride (AlPc) associated with water dispersible poly(methyl vinyl ether-co-maleic anhydride) nanoparticles is described. AlPc was associated with nanoparticles produced by a method of solvent displacement. This system was analyzed for its physicochemical characteristics, and for its photodynamic activity in vitro in cancerous (murine mammary carcinoma cell lineage 4T1, and human mammary adenocarcinoma cells MCF-7) and noncancerous (murine fibroblast cell lineage NIH/3T3, and human mammary epithelial cell lineage MCF-10A) cell lines. Cell viability and the elicited mechanisms of cell death were evaluated after the application of photodynamic therapy. This system showed improved photophysical and photochemical properties in aqueous media in comparison to the free photosensitizer, and it was effective against cancerous cells in vitro.

Thalidomide promotes leukocytosis in mice inoculated with 4T1 mammary carcinoma

The aim of this study was to evaluate the therapy effect of thalidomide in the 4T1 murine mammary carcinoma. 4T1 cell suspension was injected into the posterior left flank of all animals to obtain solid tumors. Five days after inoculation, the treatment group was orally administered 150 mg/kg of thalidomide for seven days. Tumors were measured every 48 hours until the end of treatment. Whole blood was collected for hematology analysis. Our results suggest that thalidomide therapy increase the number of circulating leukocytes in the 4T1 murine mammary carcinoma, and this response is accompanied by a decrease in tumor growth.

Integrin α3β1 Can Function to Promote Spontaneous Metastasis and Lung Colonization of Invasive Breast Carcinoma

Significant evidence implicates α3β1 integrin in promoting breast cancer tumorigenesis and metastasis-associated cell behaviors in vitro and in vivo. However, the extent to which α3β1 is actually required for breast cancer metastasis remains to be determined. We used RNA interference to silence α3 integrin expression by approximately 70% in 4T1 murine mammary carcinoma cells, a model of aggressive, metastatic breast cancer. Loss of α3 integrin reduced adhesion, spreading, and proliferation on laminin isoforms, and modestly reduced the growth of orthotopically implanted cells. However, spontaneous metastasis to lung was strikingly curtailed. Experimental lung colonization after tail vein injection revealed a similar loss of metastatic capacity for the α3-silenced (α3si) cells, suggesting that critical, α3-dependent events at the metastatic site could account for much of α3β1's contribution to metastasis in this model. Reexpressing α3 in the α3si cells reversed the loss of metastatic capacity, and silencing another target, the small GTPase RhoC, had no effect, supporting the specificity of the effect of silencing α3. Parental, α3si, and α3-rescued cells, all secreted abundant laminin α5 (LAMA5), an α3β1 integrin ligand, suggesting that loss of α3 integrin might disrupt an autocrine loop that could function to sustain metastatic growth. Analysis of human breast cancer cases revealed reduced survival in cases where α3 integrin and LAMA5 are both overexpressed. α3 integrin or downstream effectors may be potential therapeutic targets in disseminated breast cancers, especially when laminin α5 or other α3 integrin ligands are also over-expressed.

When Seed and Soil Theory Meets Chicken or Egg Theory in Cancer Metastasis.

Cancer metastasis has been a serious problem since decades. Seed and soil hypothesis of metastasis remains true and all the metastatic tumors follow this nature’s law [1,2]. Advance metastasis or multi-organ metastasis is difficult to treat due to multi-organ dysfunction. One of the major issues in metastasis is that diagnosis occurs at the advanced stages. Secondly, we have not understood the complete mechanisms so well so far due to intricate nature of metastasis. In metastasis, seed (tumor cell) migrates to the soil (distant organs, e.g. lung, liver, brain, and bone). Several experimental studies have been done that suggested the role of bone marrow derived progenitor cells [3] (e.g. CD11b+ [4] and VEGFR1+ [5] cells) in the initiation of metastasis. Several chemokines, such as SDF-1, TNF-α, TGF-β and VEGF-A influence the recruitment of different cell types to pre-metastatic sites resulting into increased expression of specific molecules in the niche like S100A8, S100A9, lysyl oxidase (LOX), fibronectin, MMP9 and MMP2 in the initiation of premetastatic niche [6,7], which are bonafide candidates of therapeutic targeting [8]. In addition, tumor induced hypoxia has been shown to promote the premetastatic niche formation by recruiting CD11b+/Ly6Cmed/Ly6G+ cells [9] and producing LOX [10]. Recently, much attention has been given to the tumor-derived exosomes or micro vesicles that carry almost every essential cellular macromolecule and has signals to polarize cells in the tumor microenvironment and create premetastatic niche in the distant organs, before the seed (tumor cell) arrives [11,12]. Exosomes derived from melanomas were shown to educate pro-metastatic progenitor cells in the bone marrow [13]. Renal-carcinoma-derived exosomes were found to promote angiogenesis in lung tumor metastases [14]. In addition, using murine mammary carcinoma demonstrated that, tumor-derived microvesicles use osteopontin to mobilize pro-angiogenic cells from the bone marrow [15]. Surprisingly, exosomes perform cell independent miRNA biogenesis to promote tumorigenesis and metastasis [16]. Firstly, tumor derived exosomes has pro-angiogenic functions that helps tumor in building required vasculature for tumor growth. For example, Yoon et al. [17] investigated pro-angiogenic role of tumor-secreted exosomes by showing Egr-1 activation in endothelial cells through ERK1/2 and JNK signaling pathways and endothelial cell migration, which was facilitated by the tumor cell derived extracellular vesicles. On the other hand, tumor derived exosomes involved in the destruction of vasculature integrity for metastasis. For example, miR-105, which is characteristically expressed and secreted by metastatic breast cancer cells, is reported as a potent regulator of tumor cell migration through targeting the tight junction protein ZO-1 via exosomes. Tumor cell secreted exosomes deliver miR-105 to the site of endothelial monolayers that efficiently destroys tight junctions and hence the integrity of barriers against metastasis [18]. Although exosomes have attracted much attention and are considered as a bonafide targets for cancer therapy, their roles in tumor metastasis is poorly investigated. In addition, technologies and methods to study exosomes are growing day by day. It is possible that tumor cell exosomes are delivered to the distant organs that manipulates host environment before any immune cells or chemokine. However, what initiates tumor cell migration to the distant organs, remains unclear, i.e. chicken comes first or egg and warrants further investigations.

Vascular Shutdown and Pro-inflammatory Cytokine Expression in Breast Cancer Tumors after Photodynamic Therapy Mediated by Nano-sized Liposomes Containing Aluminium-Chloride-Phthalocyanine

Breast cancer is the most prevalent solid tumor worldwide, being the second most common cause of death in women. Treatments recommended for breast cancer include both local and regional protocols such as surgery and radiotherapy, and systemic procedures, such as chemotherapy and hormonal therapies. In most of them, there is much functional impairment and low selectivity for tumor, leading to immunosuppression. Alternatively, some new protocols such as Photodynamic therapy (PDT) has been proposed as a therapeutic option for breast cancer management. One of the promising features related to PDT is immune activation against tumor cells. Thus, considering the importance of immunological activation against tumor cells after PDT, the aim of this study was to evaluate the expression of local and systemic inflammatory cytokines in breast cancer-bearing mice after PDT application, mediated by the photosensitizer aluminium-chlorine-phtalocyanine (AlClPc) in liposomal formulation (Lipo-AlClPc). In addition, we evaluate the efficacy of this PDT protocol in the treatment of murine mammary carcinoma cell line – 4T1 in in vitro and in vivo experimental models. It could be observed in the present study that PDT mediated by local application of Lipo- AlClPc in the treatment of breast cancer in Balb / c mice acted by multiple mechanisms, including the induction of cell death by necrosis and due to direct oxidative damage to tumor cells and vascular shutdown. All these events contributed to the expression of inflammatory cytokines, resulting in a subsequent anti-tumor inflammatory response. In conclusion, PDT mediated by Lipo-AlClPc is capable of activating an inflammatory response, providing support to be used at least as an adjunct therapy to surgical treatments, allowing greater tumor control for long periods and, possibly, greater control of metastatic foci.

Macrophages Are More Potent Immune Suppressors Ex Vivo Than Immature Myeloid-Derived Suppressor Cells Induced by Metastatic Murine Mammary Carcinomas

Myeloid-derived suppressor cells (MDSCs) are emerging as potential promoters of metastatic tumor growth, and there is interest in targeting immature MDSCs by inducing their differentiation into more mature myeloid cells. We used all-trans retinoic acid (ATRA) to differentiate MDSCs in mice bearing metastatic 4T1 or 4TO7 murine mammary tumors, and assessed the immune-suppressive mechanisms and potencies of different myeloid cell subpopulations. Metastatic mammary tumors induced the accumulation of distinct populations of immature CD11b(+)Gr1(+)F4/80(-)Ly6C(mid)Ly6G(+) MDSCs ("Gr1(+) cells") and mature CD11b(+)Gr1(-)F4/80(+) cells ("F4/80(+) cells") in metastatic target organs. ATRA triggered the differentiation of Gr1(+) cells into F4/80(+) cells in the lungs and, unexpectedly, enhanced pulmonary metastatic tumor growth. We found that F4/80(+)Ly6C(-)Ly6G(-) mature macrophages (Ms) were up to 30-fold more potent immune suppressors than Gr1(+) cells on a per-cell basis, which we postulate may contribute to the increased metastatic growth observed with ATRA treatment. F4/80(+) cells and Gr1(+) cells used different reactive oxygen species (ROS)-mediated mechanisms of immunosuppression ex vivo, with F4/80(+) cells producing higher levels of ROS, which is consistent with their superior immunosuppressive abilities. These data highlight the potent immunosuppressive functions of Ms, reveal that Ms can suppress T cell responses via ROS production, and suggest that ROS inhibitors may be useful in promoting antitumor immune responses. Our findings also caution against using ATRA to modulate myeloid cell differentiation and function to treat breast cancer metastases in the lung, and support the development of therapeutic strategies to enhance antitumor immunity by targeting myeloid cells as a collective group.

Osteolysis and pain due to experimental bone metastases are improved by treatment with rapamycin

In advanced breast cancer, bone metastases occur in 70 % of patients. Managing the devastating pain associated with the disease is difficult. Rapamycin is an immunomodulatory drug that targets the mammalian target of rapamycin pathway. Rapamycin has been shown to decrease osteolysis associated with metastatic breast cancer in pre-clinical models and to reduce pain in inflammatory and neuropathic models. The aim of this study was to evaluate the effectiveness of rapamycin in reducing pain associated with experimental osteolytic metastases. Bone cancer was induced by intra-tibial injections of murine mammary carcinoma cells (4T1) in immunocompetent BALB/c mice and treated intraperitoneally for up to 5 weeks with vehicle, rapamycin or pamidronate (a bisphosphonate currently used to reduce bone loss in bone cancer patients). The control group received intra-tibial injection with saline (sham) and was treated with vehicle intraperitoneally. Cancer-induced osteolysis was observed histologically and radiographically 2-3 weeks following cancer inoculation and gradually increased with time. Measures of evoked nociceptive behaviors including sensitivity to mechanical, thermal, and cold stimuli and spontaneous nociceptive behaviors (limping, guarding) were evaluated. Significant hypersensitivity to sensory stimuli developed in cancer-bearing mice compared to sham 3 weeks following inoculation. Rapamycin decreased or delayed the development of cancer-induced mechanical, heat, and cold hypersensitivity, while pamidronate reduced heat and cold hypersensitivity. Both rapamycin and pamidronate had a partial protective effect on the spontaneous nociceptive behaviors, limping and guarding. Our data suggest that rapamycin may have efficacy in the management of pain associated with metastatic breast cancer.

Mitogen-activated Protein Kinase Signaling Mediates Phosphorylation of Polycomb Ortholog Cbx7

Cbx7 is one of five mammalian orthologs of the Drosophila Polycomb. Cbx7 recognizes methylated lysine residues on the histone H3 tail and contributes to gene silencing in the context of the Polycomb repressive complex 1 (PRC1). However, our knowledge of Cbx7 post-translational modifications remains limited. Through combined biochemical and mass spectrometry approaches, we report a novel phosphorylation site on mouse Cbx7 at residue Thr-118 (Cbx7T118ph), near the highly conserved Polycomb box. The generation of a site-specific antibody to Cbx7T118ph demonstrates that Cbx7 is phosphorylated via MAPK signaling. Furthermore, we find Cbx7T118 phosphorylation in murine mammary carcinoma cells, which can be blocked by MEK inhibitors. Upon EGF stimulation, Cbx7 interacts robustly with other members of PRC1. To test the role of Cbx7T118 phosphorylation in gene silencing, we employed a RAS-induced senescence model system. We demonstrate that Cbx7T118 phosphorylation moderately enhances repression of its target gene p16. In summary, we have identified and characterized a novel MAPK-mediated phosphorylation site on Cbx7 and propose that mitogen signaling to the chromatin template regulates PRC1 function.

Abstract C1: Functional normalization of the breast cancer vasculature through activation of Tie2 using a vascular-endothelial protein tyrosine phosphatase inhibitor.

Abstract Background: The solid tumor microvasculature is characterized by structural immaturity and functional abnormality, and mediates several deleterious aspects of tumor behavior. The vascular endothelial protein tyrosine phosphatase (VE-PTP) attenuates the activity of the endothelial cell (EC) Tie-2 receptor tyrosine kinase, a key mediator of vessel maturation. Here we determine the role of VE-PTP in the vasculature of primary and metastatic mammary carcinomas. Methods: AKB-9778 is a first-in-class pharmacologic VE-PTP inhibitor. We systematically examined its effects in vitro and in vivo. First we studied the effects of AKB-9778 on Tie-2 signaling in two endothelial cell lines in vitro, and also measured its impact on Tie-2 activation in normal and tumor ECs in mice. We next used embryonic zebrafish assays to determine the effects of AKB-9778 on embryonic angiogenesis. We also studied the impact of AKB-9778 therapy on the tumor vasculature, tumor growth and metastatic progression using orthotopic models of murine mammary carcinoma as well as spontaneous and experimental metastasis models (4T1, E0771, P0008, MMTV-PyMT). Finally, we used endothelial nitric oxide synthase (eNOS) deficient mice to establish the role of eNOS in mediating the effects of VE-PTP inhibition. Results: AKB-9778 induced ligand-independent Tie-2 activation in ECs in vitro and in vivo, and impaired embryonic zebrafish angiogenesis. In mouse models of breast cancer, AKB-9778 (i) delayed the early phase of mammary tumor growth by maintaining vascular maturity; (ii) slowed the growth of spontaneous micrometastases by preventing extravasation of tumor cells into distant organ parenchyma (prolonging survival above adjuvant chemotherapy alone); and (iii) matured established primary tumor blood vessels (increased pericyte coverage, reduced permeability) in turn enhancing tumor perfusion, reducing hypoxia, and improving radiation response. Experiments using eNOS knockout mice showed that the effects of AKB-9778 on tumor vessels were mediated in part by eNOS activation. Conclusions: Phosphatase inhibition is a relatively unexplored field in cancer, and this is to our knowledge the first demonstration of the effects of VE-PTP inhibition in any disease. Our results demonstrate that pharmacological VE-PTP inhibition can normalize the structure and function of tumor vessels through Tie-2 activation, which delays tumor growth, slows metastatic progression, and enhances response to concomitant cytotoxic treatments. Furthermore we provide genetic evidence for the causal role of eNOS in mediating the beneficial effects of VEPTP inhibition in established tumors. By using models of adjuvant therapy (combined with chemotherapy) and radiation treatment, our results indicate possible avenues for further studying the clinical translatability of VE-PTP inhibition in cancer. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C1. Citation Format: Shom Goel, Nisha Gupta, Brian P. Walcott, Matija Snuderl, Cristina T. Kesler, Benjamin J. Vakoc, Randall T. Peterson, Timothy P. Padera, Dan G. Duda, Dai Fukumura, Rakesh K. Jain. Functional normalization of the breast cancer vasculature through activation of Tie2 using a vascular-endothelial protein tyrosine phosphatase inhibitor. [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 C1.

Abstract A256: Epigenetic priming with novel hypomethylating agent SGI-110 improved antitumor activity of CTLA-4 blockade in a syngenic mouse model.

Abstract Background. The second generation DNA hypomethylating agent SGI-110, a dinucleotide of decitabine (DAC) and deoxyguanosine formulated as a small volume SQ injection allowing extended DAC exposure compared to DAC i.v., has shown promising anti-cancer clinical activity. Our recent pre-clinical in vitro and in vivo data provides evidence that SGI-110 bears immunomodulating activities which improve the recognition of neoplastic cells by cytotoxic T lymphocytes. In addition, the hypomethylating activity of SGI-110 on cancer testis antigens (CTA)-specific promoters and the resulting induction/up-regulation of CTA expression have been extensively characterized in PBMCs from MDS or AML patients enrolled in a randomized Phase 1-2 First in Human PK/PD-guided, dose-escalation study. These well-defined immunomodulatory properties of SGI-110 prompt its potential use for the development of new immunotherapeutic approaches. Accordingly, in this study, we explored whether SGI-110 might improve the clinical efficacy of an anti-CTLA-4 monoclonal antibody (mAb). Materials and Methods. Balb/c mice (6/group) were injected SQ in the flank region with murine mammary carcinoma cells TS/A (2×105). Mice bearing palpable tumor grafts (diameter ≥ 0.2 cm) were treated with 3mg/kg of reconstituted SGI-110 (daily x 5) from day 1, alone or in combination with 100µg/hamster anti-murine CTLA-4 mAb concomitant with (days 2, 5 and 8) or subsequent to (days 8, 11 and 14) SGI-110. Control mice were injected with diluent for reconstitution. The effectiveness and tolerability of treatment was evaluated by tumor volume and body weight measurements, respectively. Results. The best antitumor effect was achieved in mice treated with SGI-110 followed by anti-CTLA-4 mAb; in detail, a tumor mass significantly (p&amp;lt;0.05) smaller than that of control mice was observed, indicating, at day 26, a tumor growth inhibition of 84.4%. Moreover, a significant, but lower, reduction in tumor mass occurred in SGI-110-treated mice as compared to control mice, with a tumor growth inhibition of 62.9% at days 26. No difference was observed in tumor growth inhibition in mice treated with SGI-110 combined with concomitant anti-CTLA-4 mAb treatment as compared to mice treated with SGI-110 alone. Noteworthy, no loss in body weight was observed in all mice investigated, demonstrating a good tolerability of therapeutic regimens tested. Conclusion. Altogether, these data provide evidence for a scientific rationale to develop novel and more effective epigenetic-immunotherapeutic strategies for cancer patients. Accordingly, an exploratory phase I clinical trial to evaluate safety and immunobiologic activities of epigenetic priming with SGI-110 followed by CTLA-4 blockade is being planned. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A256. Citation Format: Alessia Covre, Carolina Fazio, Hugues Nicolay, Giulia Parisi, Pietro Taverna, Mohammad Azab, Sandra Coral, Michele Maio. Epigenetic priming with novel hypomethylating agent SGI-110 improved antitumor activity of CTLA-4 blockade in a syngenic 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 A256.

Abstract B126: Antiprogestins induce tumor vessels remodeling and increase the effects of albumin-bound paclitaxel or doxorubicin-pegylated liposomes in PR-A (+) but not in PR-B (+) T47D xenografts

Abstract Chemotherapy is the standard treatment for metastatic breast cancer today. To overcome undesired side effects and improve drug efficacy in tumor cells, different nanoparticle formulations were developed. Paclitaxel was bound with human albumin (Nab-paclitaxel; Abraxane), and pegylated liposomes have been developed for doxorubicin delivery (PEG-LD; Doxopeg). We have evaluated the effect of these therapeutic agents together with antiprogestins in experimental murine mammary carcinomas (MMC) expressing different ratios of progesterone receptor (PR) isoforms and showed an increased therapeutic effect of combined treatments vs. single treatments only in tumors over expressing PR isoform A (PR-A). We showed that in these tumors antiprogestins such as Mifepristone (MFP) or Proellex, induced tissue remodeling increasing the stroma/parenchyma ratio and the number of vessels, increasing doxorubicin or Abraxane concentration within the tumors. (Sequeira et al., CTRC-AACR San Antonio Breast Cancer Symposium, December 4-8 2012). The aims of this study were: a) to characterize the tumor vessels generated during antiprogestin treatment in the MMC and b) to evaluate the effect of chemotherapeutic agents together with MFP vs. single treatments, on the growth of human T47D breast cancer cells expressing PR-A or PR isoform B (PR-B), inoculated orthotopically into NOD/LtSz-scid/IL-2Rgamma null female mice. BALB/c female mice bearing 59-2-HI (PR-A&amp;gt;PR-B) tumors on one flank and C4-2-HI (PR-B&amp;gt;PR-A) on the other flank were treated for 5 days with MFP (10 mg/kg/day). Mice were then injected with Fluorescein labeled Lycopersicon Esculentum (Tomato) Lectin (TL), or with Count Control Beads (CCB) and sacrificed after 7 min or 60 min respectively. Immunofluorescence microscopy studies showed increased TL staining (p&amp;lt;0.05), and vessels with increased diameter (p&amp;lt;0.001) in MFP-treated tumors showing higher levels of PR-A than PR-B as compared to untreated mice. Similarly, higher levels of fluorescent CCB/total number of cells were detected by flow cytometry in the same experimental groups suggesting that an increased vascular network is responsible, at least partially, of increasing the nano-chemotherapeutic drug concentration within these tumors. On the other hand, the T47D-YA and T47D-YB xenografts showed a similar responsiveness as the MMC: PEG-DL (0.9 mg/Kg/week) or Abraxane (10 mg/Kg; three doses every 4 days) combined with MFP (10 mg/Kg/Day) were more effective than the single treatments decreasing tumor size, only in the T47D-YA xenografts (p&amp;lt;0.05). We propose that antiprogestins have the potential to enhance the efficacy of nano-chemotherapeutic formulations in patients with breast cancer showing higher levels of PR-A than PR-B by increasing their access to the tumor cells. Citation Format: Gonzalo R. Sequeira, Silvia I. Vanzulli, Maria May, Paola Rojas, Caroline A. Lamb, Alfredo Molinolo, Claudia Lanari. Antiprogestins induce tumor vessels remodeling and increase the effects of albumin-bound paclitaxel or doxorubicin-pegylated liposomes in PR-A (+) but not in PR-B (+) T47D xenografts. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr B126.

Abstract A109: A signal peptide as a putative tool against MMTV-associated breast cancers

Abstract Mouse Mammary Tumor Virus (MMTV) causes mammary carcinoma and lymphoma in mice. An increasing body of evidence from different laboratories suggests an association between MMTV and up to 38% of human breast cancers. This seems related to severity of the disease and geographical location. Signal peptides are N-terminal extensions of nascent secretory and membrane proteins (typically including 15-25 amino acid residues). They mediate translocation across, or insertion into the membrane of the endoplasmic reticulum (ER). The signal peptide of Mouse Mammary Tumor Virus (MMTV) envelop precursor (named MMTV-14 or p14 for short), in addition to being unusually long (98 a.a) demonstrates some unique characteristics: 1) It localizes to nucleoli; 2) It functions as a nuclear export factor for intron-containing viral transcripts; 3) It can function in a pro-oncogenic or anti-oncogenic capacity depending on its phosphorylation by the kinases CK2 (at serine 65) or PKC (at serine 18), respectively. We report that, in addition to its nucleolar localization, p14 is expressed on the cell surface of murine lymphomas and mammary carcinomas that harbor MMTV, as well as on the surface of MCF-7 human breast cancer cells ectopically expressing p14. Cell surface expression allows p14 to play the role of a tumor associated antigen. Indeed, priming and boosting of mice with recombinant p14, using different adjuvants, induces specific anti-p14 antibodies, as well as immunizes these mice against malignant tumor cells that harbor MMTV. This immunity is adoptively transferred to naïve mice. Thus, MMTV-p14 is a multi-faceted signal peptide with immune modulating characteristics that can be used for developing both preventive and therapeutic approaches towards MMTV-associated breast cancers. Supported in part by a Kamin grant from the Office of the Chief Scientist. Citation Format: Ori Braitbard, Allan Bar-Sinai, Maayan Roniger, Dana Rajchman, Nira Hochman, Jacob Hochman. A signal peptide as a putative tool against MMTV-associated breast cancers. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr A109.

Effects of Vascular-Endothelial Protein Tyrosine Phosphatase Inhibition on Breast Cancer Vasculature and Metastatic Progression

The solid tumor microvasculature is characterized by structural and functional abnormality and mediates several deleterious aspects of tumor behavior. Here we determine the role of vascular endothelial protein tyrosine phosphatase (VE-PTP), which deactivates endothelial cell (EC) Tie-2 receptor tyrosine kinase, thereby impairing maturation of tumor vessels. AKB-9778 is a first-in-class VE-PTP inhibitor. We examined its effects on ECs in vitro and on embryonic angiogenesis in vivo using zebrafish assays. We studied the impact of AKB-9778 therapy on the tumor vasculature, tumor growth, and metastatic progression using orthotopic models of murine mammary carcinoma as well as spontaneous and experimental metastasis models. Finally, we used endothelial nitric oxide synthase (eNOS)-deficient mice to establish the role of eNOS in mediating the effects of VE-PTP inhibition. All statistical tests were two-sided. AKB-9778 induced ligand-independent Tie-2 activation in ECs and impaired embryonic zebrafish angiogenesis. AKB-9778 delayed the early phase of mammary tumor growth by maintaining vascular maturity (P < .01, t test); slowed growth of micrometastases (P < .01, χ(2) test) by preventing extravasation of tumor cells (P < 0.01, Fisher exact test), resulting in a trend toward prolonged survival (27.0 vs 36.5 days; hazard ratio of death = 0.33, 95% confidence interval = 0.11 to 1.03; P = .05, Mantel-Cox test); and stabilized established primary tumor blood vessels, enhancing tumor perfusion (P = .03 for 4T1 tumor model and 0.05 for E0771 tumor model, by two-sided t tests) and, hence, radiation response (P < .01, analysis of variance; n = 7 mice per group). The effects of AKB-9778 on tumor vessels were mediated in part by endothelial nitric oxide synthase activation. Our results demonstrate that pharmacological VE-PTP inhibition can normalize the structure and function of tumor vessels through Tie-2 activation, which delays tumor growth, slows metastatic progression, and enhances response to concomitant cytotoxic treatments.

Increased expression and copy number amplification of LINE-1 and SINE B1 retrotransposable elements in murine mammary carcinoma progression

In higher eukaryotic genomes, Long Interspersed Nuclear Element 1 (LINE-1) retrotransposons and endogenous retroviruses represent large families of repeated elements encoding reverse transcriptase (RT) proteins. Short Interspersed Nuclear Element B1 (SINE B1) retrotrasposons do not encode RT, but use LINE-1-derived RT for their retrotransposition. We previously showed that many cancer types have an abundant endogenous RT activity. Inhibition of that activity, by either RNA interference-dependent silencing of active LINE-1 elements or by RT inhibitory drugs, reduced proliferation and promoted differentiation in cancer cells, indicating that LINE-1-encoded RT is required for tumor progression. Using MMTV-PyVT transgenic mice as a well-defined model of breast cancer progression, we now report that both LINE-1 and SINE B1 retrotransposons are up-regulated at a very early stage of tumorigenesis; LINE-1-encoded RT product and enzymatic activity were detected in tumor tissues as early as stage 1, preceding the widespread appearance of histological alterations and specific cancer markers, and further increased in later progression stages, while neither was present in non-pathological breast tissues. Importantly, both LINE-1 and SINE B1 retrotransposon families undergo copy number amplification during tumor progression. These findings therefore indicate that RT activity is distinctive of breast cancer cells and that, furthermore, LINE-1 and SINE B1 undergo copy number amplification during cancer progression.