Polyoma Middle T Research Articles

The Synthetic Triterpenoid CDDO-Methyl Ester Delays Estrogen Receptor–Negative Mammary Carcinogenesis in Polyoma Middle T Mice

Novel drugs are needed for the prevention and treatment of breast cancer. Synthetic triterpenoids are a promising new class of compounds with activity in a variety of preclinical cancer models. We tested activity of the methyl ester derivative of the synthetic triterpenoid, 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid (CDDO-Me), in a relevant model of estrogen receptor-negative breast cancer, the polyoma-middle T (PyMT), in which the oncoprotein drives carcinogenesis. The developing tumors recapitulate key features of the human disease. Mice were fed CDDO-Me (50 mg/kg diet), starting at 4 weeks of age. CDDO-Me significantly increased the age of mice at onset of first tumor (P < 0.001) by an average of 4.3 weeks and overall survival (P < 0.001) by 5.2 weeks. The drug also inhibited the infiltration of tumor-associated macrophages into mammary glands of PyMT mice at 12 weeks of age and reduced levels of the chemokines CXCL12 and CCL2 in primary PyMT mammary tumor cells. Treatment with this multifunctional drug also inhibited secretion of matrix metalloproteinase-9 in primary tumor cells from PyMT mice and decreased proliferation of these cells by inhibiting cyclin D1 and decreasing phosphorylation of epidermal growth factor receptor and STAT3.

Abstract 3416: Ubiquitous Brms1 expression is critical for mammary carcinoma metastasis suppression via promotion of apoptosis

Abstract Morbidity and mortality in breast cancer patients are drastically increased when primary tumor cells are able to spread to distant sites and proliferate to become secondary lesions. Effective treatment of metastatic disease has been limited; therefore, increased molecular understanding to identify biomarkers and targets is needed. We have previously shown that breast cancer metastasis suppressor 1 (Brms1) can suppress development of pulmonary metastases when expressed in a variety of cancer types, including metastatic mammary carcinoma. Our lab has developed two transgenic Brms1 mouse models, one which expresses murine Brms1 cDNA specifically in mammary tissue (expression by the mouse mammary tumor virus (MMTV) promoter) and a ubiquitous Brms1 expression model (expression by the chicken beta actin promoter). The goal of this study was to investigate mechanisms of Brms1-mediated metastasis suppression in transgenic mice that express Brms1 using a polyoma middle T (PyMT) oncogene-induced model. Brms1 expression, either ubiquitously or predominantly in the mammary gland, did not significantly alter growth of the primary tumor, confirming earlier studies. When expressed ubiquitously, Brms1 suppressed pulmonary metastasis and promoted tumor cell apoptosis in the lung but not in the mammary gland. However, selective expression of Brms1 in the mammary gland using the MMTV promoter did not significantly block metastasis nor did it promote apoptosis in mammary glands or lungs despite increased expression within primary tumors and the lungs. These results suggest tissue- or cell-type specific expression of Brms1 is a critical determinant for Brms1-mediated metastasis suppression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3416. doi:1538-7445.AM2012-3416

Metastasis is an early event in mouse mammary carcinomas and is associated with cells bearing stem cell markers

IntroductionIt is still uncertain whether metastasis is predominantly an early or late event in tumor progression. The detection of early metastases and cells responsible for the dissemination may therefore have significant clinical implications.MethodsLung dissemination and/or metastasis were investigated in mice carrying the polyomavirus middle-T oncogene (PyMT) during different stages of mammary tumorigenesis using the colony forming assay. Immunocytochemical or immunohistochemical staining was used to identify subpopulations of cells responsible for lung dissemination and metastasis. Histological examination was used to show primary and metastatic tumors. The tumor-initiating and metastatic capacity of cells expressing stem cell markers was assessed in syngeneic wild-type (WT) mice whose mammary fat pads were injected with these cells.ResultsMetastatic mammary epithelial cells were detected in the lungs of mice carrying the PyMT oncogene (MMT mice). These cells were observed early in breast tumorigenesis when the mammary tree appeared by histological inspection to be normal (or at a premalignant stage), suggesting the possession of disseminating and metastatic capacity even before full malignant transformation. Some of the disseminated cells and lung metastases displayed surface stem cell markers. These findings suggest that stem cells from apparently precancerous primary lesions could be a source of metastasis. Indeed, injection of lung tissue cells from MMT mice into syngeneic WT mice resulted in the formation of mammary tumors. These tumors resembled their parent mammary tumors in the MMT donors as well as grafted tumors derived from mammary tumor cells. Furthermore, when we injected lung tissue cells from GFP MMT mice into the fat pads of recipient WT mice, disseminated or metastatic GFP-expressing cells were detected in the lungs, lymph nodes and blood of the recipient WT mice. We finally identified a subpopulation of mammary epithelial/tumor cells expressing CD44 and Sca1 that was largely responsible for dissemination and metastasis in MMT mice.ConclusionsThe tumorigenic and metastatic potential of a subpopulation of mammary epithelial/tumor cells in MMT mice is endowed relatively early in mammary neoplasms and suggests a potential role for cancer stem cell sub-populations in metastasis.

Abstract A6: microRNA sequencing of AKXD recombinant inbred panel identifies miR-216b as a candidate metastasis suppressor in a murine model of breast cancer

Abstract Metastatic disease continues to be the primary cause of mortality in cancer. Using a combination of complex genetics, mouse models of breast cancer, and human epidemiology, our laboratory has demonstrated that germline polymorphism contributes to an individual's susceptibility to metastasis. In the current study, we have used the AKXD recombinant inbred panel, which is derived from inbred mice of metastasis resistant, DBA, and metastasis-prone, AKR, genetic backgrounds, to identify microRNAs whose expression is associated with metastasis. microRNA sequencing of tumor tissue from the progeny of AKXD x Polyoma Middle-T mice demonstrated that the expression of the miR-216/217 cluster is significantly negatively correlated with pulmonary metastases in the AKXD panel. Linkage analysis demonstrated a significant association between the miR-216/217 locus and the DBA allele, suggesting that the difference in expression is inherited rather than somatically acquired. Finally, we show that ectopic expression of miR-216b in murine mammary tumor cells suppresses pulmonary metastasis without significantly impacting primary tumor growth in vivo. These findings suggest that miR-216b is an inherited modifier of metastasis with metastasis suppressing activity in a mouse model of breast cancer. Citation Format: Farhoud Faraji, Ying Hu, Natalie Goldberger, Gang Wu, Ken H. Buetow, Jinghui Zhang, Kent W. Hunter. microRNA sequencing of AKXD recombinant inbred panel identifies miR-216b as a candidate metastasis suppressor in a murine model of breast cancer [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer; 2012 Jan 8-11; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(2 Suppl):Abstract nr A6.

Ospemifene and 4-hydroxyospemifene effectively prevent and treat breast cancer in the MTag.Tg transgenic mouse model

Ospemifene, a new drug indicated for the treatment of vulvovaginal atrophy, has completed phase III clinical trials. A condition affecting millions of women worldwide, vulvovaginal atrophy has long been treated with estrogen therapy. Estrogen treatment carries with it risks of thromboembolism, endometrial proliferative effects, and breast cancer promotion. In this study, we test the effects of three dosing levels of ospemifene in both the prevention and treatment of breast cancer in the MTag.Tg mouse model. The polyomavirus middle-T transgenic mouse model (MTag.Tg), which produces synchronized, multifocal mammary tumors in the immunologically intact C57BL/6 background, was used to examine the impact of ospemifene treatment. First, a cell line derived from an MTag.Tg mouse tumor (MTag 34) was treated in vitro with ospemifene and its major metabolite, 4-hydroxyospemifene (4-OH ospemifene). MTag.Tg mice were treated daily by gavage with three different doses of ospemifene (5, 25, and 50 mg/kg) before or after the development of mammary tumors. Survival and tumor development results were used to determine the effect of ospemifene treatment on mammary tumors in both the preventive and treatment settings. Tumors and the MTag 34 cell line were positive for estrogen receptor expression. The MTag 34 line was not stimulated by ospemifene or its major, active metabolite 4-OH ospemifene in vitro. Ospemifene increased survival time and exerted an antitumor effect on the development and growth of estrogen receptor-positive mammary tumors in the MTag.Tg mouse model at the 50-mg/kg dose. The levels of ospemifene and 4-OH ospemifene in both the tumors and plasma of mice confirmed the dosing. Ospemifene did not exert an estrogenic effect in the breast tissue at doses equivalent to human dosing. Ospemifene prevents and treats estrogen receptor-positive MTag.Tg mammary tumors in this immune-intact mouse model in a dose-dependent fashion. Ospemifene drug levels in the plasma of treated mice were comparable with those found in humans. Combined with our previous data, ospemifene does not seem to pose a breast cancer risk in animals and slows down cancer development and progression in the MTag.Tg model.

P2-03-01: Genetic Reduction of Circulating Insulin-Like Growth Factor (IGF)-1 Differentially Impacts the Effects of Diet-Induced Obesity and Calorie Restriction on Mammary Tumor Progression.

Abstract Background: Epidemiological evidence suggests that obesity worsens breast cancer prognosis in pre- and postmenopausal women. Previously we demonstrated that diet-induced obese (DIO) mice and rats have higher circulating levels of IGF-1 and enhanced mammary tumor burden compared to normoweight controls. In contrast, we have found in several mouse models that a 30% calorie restricted (CR) diet regimen reduced serum IGF-1 levels and decreased mammary tumor growth. The present study investigated the effects of CR and DIO on mammary tumor growth in mice with normal serum levels of IGF-1 (wild-type) or mice with reduced circulating levels of IGF-1 (LID). Our goal was to identify new targets and strategies (with a focus on the IGF-1 pathway) for breaking the obesity-breast cancer link, which is important given the high prevalence of obesity in the U.S., and the strong association between obesity and breast cancer. Materials and Methods: Virgin female liver-specific IGF-1-deficient (LID) and wild-type (FVB/NCr) mice (10-12 weeks of age) were randomized (n=10-14 per group) to receive: control diet (modified AIN-76A), a 30% CR regimen, or a DIO regimen (60 kcal% fat diet). Mice consumed the experimental diets for 11 to 15 weeks prior to orthotopic injection (4th mammary fat pad) of Met-1fvb2murine mammary tumor cells derived from a polyoma middle-T antigen transgenic mouse mammary tumor. Transplanted tumors were allowed to grow for 4 weeks and tumor volume was measured by electronic calipers biweekly. Mice were then killed, serum was collected and stored at −80C, and tumors were excised, weighed and either formalin-fixed, paraffin-embedded or flash-frozen. Results: Using the LID mouse model of IGF-1 deficiency, this study established that decreased circulating IGF-1 (relative to wildtype mice) dramatically reduces Met-1fvb2 mammary tumor growth regardless of diet treatment (P=0.034). We also showed in LID mice that CR (44 +/− 15.6 mm3), relative to control diet (89 +/− 33 mm3) has no significant effect on Met-1fvb2 tumor growth, suggesting nearly all of the suppressive effects of CR on tumor growth (at least in the Met-1fvb2 model), can be explained by reduced systemic IGF-1. In contrast, while most of the DIO effect on tumor growth was ablated in the LID mice (332 +/− 104 mm3) vs. wild-type mice (3130 +/− 421 mm3), tumor growth in LID/DIO mice was significantly higher than LID/control mice (89 +/− 33, P=0.006) despite no change in IGF-1 levels, suggesting that a mechanism independent of IGF-1 contributes to some of the effects of DIO on Met-1fvb2 mammary tumor growth. Within LID mice, serum insulin, resistin and adiponectin were not modified by the CR or DIO diet. Discussion: We conclude that components of the IGF-1 pathway represent promising mechanistic targets for mimicking the anticancer effects of CR and breaking the obesity-breast cancer link. However, there are likely other mechanisms independent of IGF-1 that contribute to the breast tumor enhancing effects of DIO and that need to be defined. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-03-01.

Significant impact of different oxygen breathing conditions on noninvasive in vivo tumor-hypoxia imaging using [¹⁸F]-fluoro-azomycinarabino-furanoside ([¹⁸F]FAZA).

Background[18F]FAZA is a PET biomarker with great potential for imaging tumor hypoxia. Aim of our study was to compare [18F]FAZA uptake in mice with subcutaneous exogenous CT26 colon carcinomas and endogenous polyoma middle-T (PyV-mT) mammary carcinomas and to analyze the influence of different breathing protocols in CT26 colon carcinomas as well as the reversibility or irreversibility of [18F]FAZA uptake.MethodsWe injected subcutaneous CT26 colon carcinoma or polyomavirus middle-T (PyV-mT) mammary carcinoma-bearing mice intravenously with18F-FAZA and performed PET scans 1-3 h post injection (p.i.). To analyze the impact of oxygen supply in CT26 carcinomas we used three different breathing protocols: (P0) air; (P1) 100% oxygen 1 h prior injection until 3 h p.i.; (P2) 100% oxygen breathing starting 2 min prior tracer injection until 1 h p.i. and during the PET scans; mice were breathing air between the 2 h and 3 h 10 min static scans. Normalized PET images were analyzed by using defined regions of interest. Finally, some mice were dissected for pimonidazole immunohistochemistry.ResultsThere was no difference in18F-FAZA uptake 1-3 h p.i. between the two carcinoma types (CT26: 1.58 ± 0.45%ID/cc; PyV-mT: 1.47 ± 0.89%ID/cc, 1 h p.i., tumor size < 0.5 cm3). We measured a significant tracer clearance, which was more pronounced in muscle tissue (P0). The [18F]FAZA tumor-to-muscle-ratios in CT26 colon carcinoma-bearing mice 2 h and 3 h, but not 1 h p.i. were significantly higher when the mice breathed air (P0: 3.56 ± 0.55, 3 h) compared to the oxygen breathing protocols (P1: 2.45 ± 0.58; P2: 2.77 ± 0.42, 3 h). Surprisingly, the breathing protocols P1 and P2 showed no significant differences in T/M ratios, thus indicating that the crucial [18F]FAZA uptake phase is during the first hour after [18F]FAZA injection. Importantly, the muscle clearance was not affected by the different oxygen breathing conditions while the tumor clearance was lower when mice were breathing air.ConclusionExogenous CT26 colon carcinomas and endogenous polyoma middle-T (PyV-mT) mammary carcinomas showed no differences in [18F]FAZA uptake 1-3 h p.i. Our analysis using various breathing protocols with air (P0) and with pure oxygen (P1, P2) clearly indicate that [18F]FAZA is an appropriate PET biomarker for in vivo analysis of hypoxia revealing an enhanced tracer uptake in tumors with reduced oxygen supply. [18F]FAZA uptake was independent of tumor-type.

Abstract A205: Targeting caspase-9/PP2A interaction as a new antitumor strategy.

Abstract Background: Dysfunction in the regulation of apoptosis is associated with important pathologies, including cancers. While apoptosis is known to rely on the Bcl-2 family members and caspases, recent data suggest that the serine/threonine phosphatase PP2A is a key actor in regulating both Bcl-2 family members and caspases for cell life or cell death decision. Cell penetrating peptides are molecules that can translocate into cells without causing membrane damage, leading to their proposed use as vector for delivering therapeutic cargo. Focusing on both caspase-9 and PP2A, our goal was to develop cell penetrating peptides specifically designed against the caspase-9/PP2A interaction and evaluate their in vitro and in vivo therapeutic potential in various cancer cells and tumors. Materials and Methods: We have published the Drug Phosphatase Technology (DPT) based on the design of cell penetrating peptides obtained by association of a cell permeable shuttle, DPT-sh1 to a sequence of interest. We have also shown a direct interaction of human caspase-9/human PP2Ac, detected by co-immunoprecipitation and confirmed by far western. We decided to generate a peptide containing the previously published penetrating sequence (DPT-Sh1) associated to the site of interaction of human caspase-9 (DPT-C9h), in order to disturb the interaction caspase-9/PP2Ac. Using both mouse and human breast (BC) and non-small cell lung (NSCLC) cancer cells and tumors, we have then investigated the effect of DPT-C9 (specific to the sequence of the mouse caspase-9) and DPT-C9h (specific to the sequence of the human caspase-9) peptides. In in vivo experiments, DPT-C9 and DPT-C9h peptides were intraperitonealy administered at doses ranging from 1 to 50 mg/kg/day for up to 6 weeks. Results: We have demonstrated that both DPT-C9 and DPT-C9h peptides specifically blocked in vitro, in vivo, and ex vivo caspase-9/PP2A interaction, and induced caspase-9 activation and apoptosis in all tested cancer cell lines. In in vivo experiments, we have showed that DPT-C9 induced significant tumor growth inhibition (TGI) in mice bearing transgenic Polyoma Middle-T PymT mouse breast tumors and in mice bearing Kras mutated mouse NSCLC tumors. Similarly, DPT-C9h induced significant TGI in various primary human BC and NSCLC xenografts. Finally, no toxicity has been observed in treated mice, as well as no immunogenic response in immunodeficient mice. Conclusions: We have identified an interaction between caspase-9/PP2A that may be used as a new therapeutic target. Using the cell penetrating peptide DPT-C9h blocking this interaction, we have demonstrated that DPT-C9h had an impressive in vitro and in vivo therapeutic effect and therefore constitutes a new therapeutic approach of human cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A205.

Abstract A103: The synthetic triterpenoid CDDO-methyl ester targets tumor-associated macrophages to delay carcinogenesis in the PyMT model of estrogen receptor negative breast cancer

Abstract Breast cancer is the most common cancer among women in the United States. The incidence rates of breast cancer are no longer significantly declining, and approximately 40,000 women die from the disease each year. Hence, novel drugs are needed for the prevention and treatment of the disease. Synthetic triterpenoids are a promising new class of compounds with chemopreventive activity in a variety of preclinical cancer models. We tested the methyl ester derivative of the synthetic triterpenoid, 2-cyano-3, 12-dioxooleana-1,9(11)-dien-28-oic acid (CDDO-Me), in a relevant model of ER-negative breast cancer. In this mouse model, the polyoma-middle T (PyMT) oncoprotein drives carcinogenesis in the mammary gland. The developing tumors recapitulate key features of the human disease including significant infiltration of tumor-associated macrophages (TAM). Depletion of TAMs has been previously shown to delay tumor progression. PyMT mice were fed CDDO-Me (50 mg/kg diet), starting at 4 weeks of age. CDDO-Me significantly increased the age of mice at onset of first tumor by an average of 4.3 weeks (P &amp;lt; 0.001) and overall survival by 5.2 weeks (P &amp;lt; 0.001). CDDO-Me significantly (P &amp;lt; 0.05) inhibited the infiltration of TAMs into mammary glands of PyMT mice along with levels of the chemokines CXCL12 and CCL2 in primary PyMT mammary tumor cells. Treatment with CDDO-Me also inhibited secretion of MMP-9 in primary tumor cells from PyMT mice and decreased proliferation of these cells by inhibiting cyclin D1 and decreasing phosphorylation of EGFR and STAT3, key breast cancer biomarkers. Thus, CDDO-Me delayed breast carcinogenesis via inhibition of TAM infiltration and by inhibition of key pathways in breast carcinogenesis. Taken together, these findings implicate CDDO-Me as a promising chemopreventive agent for ER-negative breast cancer via inhibition of TAM infiltration and diverse oncogenic pathways. Citation Information: Cancer Prev Res 2011;4(10 Suppl):A103.

TGF-β Receptor II Loss Promotes Mammary Carcinoma Progression by Th17-Dependent Mechanisms

We report that IL-17 significantly increases the secretion of CXCL1 and CXCL5 from mammary carcinoma cells, which is downregulated by TGF-β through the type II TGF-β receptor (TβRII). Carcinoma cells with conditional knockout of TβRII (Tgfbr2(KO)) have enhanced sensitivity to IL-17a in the stimulation of chemokine secretion. During polyoma middle T (PyMT) induced tumor progression, levels of Th17 inducing cytokines TGF-β, IL-6, IL-23 were increased in PyMT/Tgfbr2(KO) tumors, which was associated with an increased number of Th17 cells. IL-17 increased the suppressive function of MDSCs on T cells through the upregulation of Arg, IDO, and COX2. Treatment of PyMT/Tgfbr2(KO) mice with anti-IL-17 Ab decreased carcinoma growth and metastatic burden. Analysis of human breast cancer transcriptome databases showed a strong association between IL-17 gene expression and poor outcome in lymph node positive, estrogen receptor negative or luminal B subtypes suggesting potential therapeutic approaches.

ErbB3 Ablation Impairs PI3K/Akt-Dependent Mammary Tumorigenesis

The ErbB receptor family member ErbB3 has been implicated in breast cancer growth, but it has yet to be determined whether its disruption is therapeutically valuable. In a mouse model of mammary carcinoma driven by the polyomavirus middle T (PyVmT) oncogene, the ErbB2 tyrosine kinase inhibitor lapatinib reduced the activation of ErbB3 and Akt as well as tumor cell growth. In this phosphatidylinositol-3 kinase (PI3K)-dependent tumor model, ErbB2 is part of a complex containing PyVmT, p85 (PI3K), and ErbB3, that is disrupted by treatment with lapatinib. Thus, full engagement of PI3K/Akt by ErbB2 in this oncogene-induced mouse tumor model may involve its ability to dimerize with and phosphorylate ErbB3, which itself directly binds PI3K. In this article, we report that ErbB3 is critical for PI3K/Akt-driven tumor formation triggered by the PyVmT oncogene. Tissue-specific, Cre-mediated deletion of ErbB3 reduced Akt phosphorylation, primary tumor growth, and pulmonary metastasis. Furthermore, EZN-3920, a chemically stabilized antisense oligonucleotide that targets the ErbB3 mRNA in vivo, produced similar effects while causing no toxicity in the mouse model. Our findings offer further preclinical evidence that ErbB3 ablation may be therapeutically effective in tumors where ErbB3 engages PI3K/Akt signaling.

Mammary epithelial-specific disruption of c-Src impairs cell cycle progression and tumorigenesis

The tyrosine kinase c-Src is activated in a large proportion of breast cancers, in which it is thought to play a key role in promoting the malignant phenotype. c-Src activity is also elevated in transgenic mouse models of breast cancer, including the widely used polyomavirus middle-T antigen (PyVmT) model, which provides an opportunity to study the importance of c-Src in mammary tumorigenesis. However, germline c-Src deletion in mammary epithelial and stromal compartments complicates the interpretation of in vivo tumorigenesis studies as a result of severe defects in mammary gland development. We have therefore engineered a mouse strain in which deletion of c-Src can be targeted to the mammary epithelium. We demonstrate that mammary epithelial disruption of c-Src impairs proliferation and tumor progression driven by PyVmT in vivo. Whereas related kinases substitute for c-Src in PyVmT signaling, c-Src ablation impairs cell cycle progression with decreased cyclin expression and elevated expression of cyclin-dependent kinase inhibitors. Our data indicate that c-Src has essential and unique functions in proliferation and tumor progression in this mouse model that may also be important in certain contexts in some human breast cancers.

Mesenchymal stem cells promote mammary cancer cell migration in vitro via the CXCR2 receptor

Bone metastasis is a common event during breast cancer progression. Recently, mesenchymal stem cells (MSCs) have been implicated in the metastasis of primary mammary cancer. Given that bone is the native environment for MSCs, we hypothesized MSCs facilitate the homing of circulating mammary cancer cells to the bone. To test this hypothesis, we examined in vitro whether bone derived MSCs from FVB mice could influence the migration of syngeneic murine mammary cancer cell lines derived from the polyoma virus middle-T (PyMT) model of mammary gland tumorigenesis. Our data show that conditioned media derived from MSCs significantly enhanced the migration of PyMT mammary cancer cell lines. Analysis of conditioned media using a cytokine array revealed the presence of numerous cytokines in the MSC conditioned media, most notably, the murine orthologs of CXCL1 and CXCL5 that are cognate ligands of the CXCR2 receptor. Further investigation identified that: (1) CXCL1, CXCL5 and CXCR2 mRNA and protein were expressed by the MSCs and PyMT cell lines and; (2) neutralizing antibodies to CXCL1, CXCL5 and CXCR2 or a CXCR2 small molecule inhibitor (SB265610) significantly abrogated the migratory effect of the MSC conditioned media on the PyMT cells. Therefore, in vitro evidence demonstrates that bone derived MSCs play a role in the migration of mammary cancer cells, a conclusion that has potential implications for breast to bone metastasis in vivo.

Abstract 2849: Tie2-expressing macrophages (TEM) depletion may enhance the clinical efficacy of combretastatin A-4-phosphate (CA-4-P)

Abstract Rationale &amp; Hypothesis: Combretastatin A-4-phosphate (CA-4-P), a tubulin-binding, vascular-disrupting agent (VDA), induces rapid and selective tumor vascular shutdown and secondary tumor cell death. However, acute revascularization contributes to treatment resistance. A group of myeloid cells in tumors, tumor associated macrophages (TAMs) are known to drive angiogenesis and tumor progression. A potently pro-angiogenic subpopulation of TAMs has also been identified – Tie2 expressing monocytes (TEMs). We hypothesize that TAMs and/or TEMs infiltrate CA-4-P-treated tumors and promote tumor recovery. Methodology, Results &amp; Conclusions: Late-stage Polyoma Middle T (PyMT) spontaneous murine breast adenocarcinomas were excised from CA-4-P- and saline-treated mice. Significant tumor necrosis was evident 24h following a single injection of 50mg/kg CA-4-P (19.37 ± 4.60%) compared with saline controls (1.76 ± 0.34%). This was accompanied by tumor infiltration of F4/80+ TAMs (5.61 ± 0.46% of tumor volume w.r.t. 0.71± 0.08% in controls). Significant increases in the number of MMP-9 expressing macrophages, primarily TEMs, were evident following CA-4-P treatment (5.72 ± 0.34% compared with 2.66 ± 0.25% in controls). TEMs were constitutive expressers of hypoxia-regulated CXCR4, which interacts via CXCL12 as a monocyte/macrophage chemoattractant. AMD3100, a chemical antagonist of CXCR4, was employed in combination with CA-4-P to prevent TEM recruitment into drug-treated tumors. TEM numbers were significantly depleted, which corresponded to heightened vascular damage (27% tumor necrosis w.r.t. 8% in CA-4-P alone). These results were replicated in a subcutaneous N202 murine mammary carcinoma model following 3 daily doses of 50mg/kg CA-4-P. Tumor growth was also significantly reduced at 72h in the combined treatment group (mean tumor volume ∼200mm3 w.r.t. ∼300mm3 in CA-4-P alone). In addition, specific TEM depletion was achieved in the N202 carcinoma model via genetic manipulation and bone marrow transplantation. Tumor necrosis was increased significantly when CA-4-P was administered in combination with TEM depletion (22.45%), compared to saline controls (0.37%) and CA-4-P therapy alone (12.89%). This was attributed to a significant reduction in MMP-9 positive TEMs (0.08 ± 0.18% compared with 1.37 ± 0.21% in saline controls and 3.67± 0.11% in CA-4-P treated tumors). Collectively, these data suggest that TEMs protect against CA-4-P or promote recovery of tumors following CA-4-P treatment. Therefore, targeting TEMs, in combination with vascular-disrupting agents such as CA-4-P may provide a novel therapeutic strategy for solid tumors. A.F. Welford was funded by a Cancer Research UK PhD studentship. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2849. doi:10.1158/1538-7445.AM2011-2849

Development of JNK2-Selective Peptide Inhibitors That Inhibit Breast Cancer Cell Migration

Despite their lack of selectivity toward c-Jun N-terminal kinase (JNK) isoforms, peptides derived from the JIP (JNK Interacting Protein) scaffolds linked to the cell-penetrating peptide TAT are widely used to investigate JNK-mediated signaling events. To engineer an isoform-selective peptide inhibitor, several JIP-based peptide sequences were designed and tested. A JIP sequence connected through a flexible linker to either the N-terminus of an inverted TAT sequence (JIP(10)-Δ-TAT(i)) or to a poly arginine sequence (JIP(10)-Δ-R(9)) enabled the potent inhibition of JNK2 (IC(50) ≈ 90 nM) and exhibited 10-fold selectivity for JNK2 over JNK1 and JNK3. Examination of both peptides in HEK293 cells revealed a potent ability to inhibit the induction of both JNK activation and c-Jun phosphorylation in cells treated with anisomycin. Notably, Western blot analysis indicates that only a fraction of total JNK must be activated to elicit robust c-Jun phosphorylation. To examine the potential of each peptide to selectively modulate JNK2 signaling in vivo, their ability to inhibit the migration of Polyoma Middle-T Antigen Mammary Tumor (PyVMT) cells was assessed. PyVMTjnk2-/- cells exhibit a lower migration potential compared to PyVMTjnk2+/+ cells, and this migration potential is restored through the overexpression of GFP-JNK2α. Both JIP(10)-Δ-TAT(i) and JIP(10)-Δ-R(9) inhibit the migration of PyVMTjnk2+/+ cells and PyVMTjnk2-/- cells expressing GFP-JNK2α. However, neither peptide inhibits the migration of PyVMTjnk2-/- cells. A control form of JIP(10)-Δ-TAT(i) containing a single leucine to arginine mutation lacks ability to inhibit JNK2 in vitro cell-free and cell-based assays and does not inhibit the migration of PyVMTjnk2+/+ cells. Together, these data suggest that JIP(10)-Δ-TAT(i) and JIP(10)-Δ-R(9) inhibit the migration of PyVMT cells through the selective inhibition of JNK2. Finally, the mechanism of inhibition of a D-retro-inverso JIP peptide, previously reported to inhibit JNK, was examined and found to inhibit p38MAPKα in an in vitro cell-free assay with little propensity to inhibit JNK isoforms.

Aligned Collagen Is a Prognostic Signature for Survival in Human Breast Carcinoma

Evidence for the potent influence of stromal organization and function on invasion and metastasis of breast tumors is ever growing. We have performed a rigorous examination of the relationship of a tumor-associated collagen signature-3 (TACS-3) to the long-term survival rate of human patients. TACS-3 is characterized by bundles of straightened and aligned collagen fibers that are oriented perpendicular to the tumor boundary. An evaluation of TACS-3 was performed in biopsied tissue sections from 196 patients by second harmonic generation imaging of the backscattered signal generated by collagen. Univariate analysis of a Cox proportional hazard model demonstrated that the presence of TACS-3 was associated with poor disease-specific and disease-free survival, resulting in hazard ratios between 3.0 and 3.9. Furthermore, TACS-3 was confirmed to be an independent prognostic indicator regardless of tumor grade and size, estrogen or progesterone receptor status, human epidermal growth factor receptor-2 status, node status, and tumor subtype. Interestingly, TACS-3 was positively correlated to expression of stromal syndecan-1, a receptor for several extracellular matrix proteins including collagens. Because of the strong statistical evidence for poor survival in patients with TACS, and because the assessment can be performed in routine histopathological samples imaged via second harmonic generation or using picrosirius, we propose that quantifying collagen alignment is a viable, novel paradigm for the prediction of human breast cancer survival.

Mena deficiency delays tumor progression and decreases metastasis in polyoma middle-T transgenic mouse mammary tumors

IntroductionThe actin binding protein Mammalian enabled (Mena), has been implicated in the metastatic progression of solid tumors in humans. Mena expression level in primary tumors is correlated with metastasis in breast, cervical, colorectal and pancreatic cancers. Cells expressing high Mena levels are part of the tumor microenvironment for metastasis (TMEM), an anatomical structure that is predictive for risk of breast cancer metastasis. Previously we have shown that forced expression of Mena adenocarcinoma cells enhances invasion and metastasis in xenograft mice. Whether Mena is required for tumor progression is still unknown. Here we report the effects of Mena deficiency on tumor progression, metastasis and on normal mammary gland development.MethodsTo investigate the role of Mena in tumor progression and metastasis, Mena deficient mice were intercrossed with mice carrying a transgene expressing the polyoma middle T oncoprotein, driven by the mouse mammary tumor virus. The progeny were investigated for the effects of Mena deficiency on tumor progression via staging of primary mammary tumors and by evaluation of morbidity. Stages of metastatic progression were investigated using an in vivo invasion assay, intravital multiphoton microscopy, circulating tumor cell burden, and lung metastases. Mammary gland development was studied in whole mount mammary glands of wild type and Mena deficient mice.ResultsMena deficiency decreased morbidity and metastatic dissemination. Loss of Mena increased mammary tumor latency but had no affect on mammary tumor burden or histologic progression to carcinoma. Elimination of Mena also significantly decreased epidermal growth factor (EGF) induced in vivo invasion, in vivo motility, intravasation and metastasis. Non-tumor bearing mice deficient for Mena also showed defects in mammary gland terminal end bud formation and branching.ConclusionsDeficiency of Mena decreases metastasis by slowing tumor progression and reducing tumor cell invasion and intravasation. Mena deficiency during development causes defects in invasive processes involved in mammary gland development. These findings suggest that functional intervention targeting Mena in breast cancer patients may provide a valuable treatment option to delay tumor progression and decrease invasion and metastatic spread leading to an improved prognostic outcome.

Enhanced Genetic Instability and Dasatinib Sensitivity in Mammary Tumor Cells Lacking NEDD9

Elevated expression of the NEDD9/HEF1/Cas-L scaffolding protein promotes tumor cell invasion and metastasis in multiple cancer cell types. Conversely, generation of mammary tumors in the mouse mammary tumor virus (MMTV)-polyoma virus middle T (PyVT) genetic model is delayed by a Nedd9(-/-) genotype. These activities arise from the role of NEDD9 in assembling complexes and supporting activity of cancer signaling proteins, including FAK, Src, Shc, and AKT, and would support evaluation of NEDD9 expression as an unambiguous biomarker for tumor aggressiveness. However, we here show that despite the initial delay in tumor growth, cells derived from MMTV-PyVT;Nedd9(-/-) tumors are characteristically hyperaggressive versus MMTV-PyVT;Nedd9(+/+) cells in anchorage-independent growth, in growth on three-dimensional matrix produced by tumor-associated fibroblasts, and in formation of tumors after mammary orthotopic reinjection and of lung metastases after tail vein injection. This reversal suggests the specific selection of MMTV-PyVT;Nedd9(-/-) cells for growth in an in vivo microenvironment. Indeed, MMTV-PyVT;Nedd9(-/-) cells have increased cell cycle, centrosomal, and mitotic defects, phenotypes compatible with the increased selection of these cells for aggressive growth. Intriguingly, in spite of their aggressive phenotype, MMTV-PyVT;Nedd9(-/-) cells persistently have low levels of Src activation and are hypersensitive to the Src kinase inhibitor dasatinib. These studies identify NEDD9 as a complex modulator of different aspects of mammary tumor growth.

Loss of ERα and FOXA1 expression in a progression model of luminal type breast cancer: Insights from PyMT transgenic mouse model

The classification of breast cancer into multiple molecular subtypes has necessitated the need for biomarkers that can assess tumor progression and the effects of chemopreventive agents on specific breast cancer subtypes. The goal of this study was to identify biomarkers whose expression are altered along with estrogen receptor α (ERα) in the polyoma middle-T antigen (PyMT) transgenic model of breast cancer and to investigate the chemopreventive activity of phenethyl isothiocyanate (PEITC). The diet of PyMT female mice was fortified with PEITC (8 mmol/kg) and the mammary streak and/or gross tumors and metastases in lungs were subjected to immunohistochemical analyses for ERα, FOXA1, and GATA-3. FOXA1 is associated with luminal type A cancers, while GATA-3 is a marker of luminal progenitor cell differentiation. In both control and PEITC-treated groups, there was a progressive loss of ERα and FOXA1 but persistence of GATA-3 expression indicating that the tumors retain luminal phenotype. Overall, the PyMT induced tumors exhibited the entire gamut of phenotypes from ERα+/FOXA1+/GATA-3+ tumors in the early stage to ERα±/FOXA1-/GATA-3+ in the late stage. Thus, PyMT model serves as an excellent model for studying progression of luminal subtype tumors. PEITC treated animals had multiple small tumors, indicating delay in tumor progression. Although these tumors were histologically similar to those in controls, there was a lower expression of these biomarkers in normal luminal cells indicating delay in tumor initiation. In in vitro studies, PEITC depleted AldeFluor-positive putative stem/progenitor cells, which may partly be responsible for the delay in tumor initiation.

Abstract 2372: The metastasis efficiency modifier ribosomal RNA processing 1 homologue (RRP1B) is a chromatin-associated factor

Abstract Previous experiments using the polyoma middle T (PyMT) mouse model of mammary tumorigenesis have demonstrated that host germline variation modulates metastasis in breast cancer. Recent work using the PyMT model has identified novel metastasis susceptibility genes, an example of which includes Rrp1b (ribosomal RNA processing 1 homolog B). Over-expression of Rrp1b in a mouse mammary tumor cell line induces a gene expression signature that predicts survival in a well characterized Dutch breast cancer dataset. Additionally, a polymorphism was identified within human RRP1B (G1421A; P436L), which is associated with differential survival in multiple breast cancer cohorts. Here we extend the analysis of RRP1B function by demonstrating that the Rrp1b activation gene expression signature accurately predicts survival in three additional publicly available breast carcinoma gene expression datasets. We have also investigated the mechanism of action of RRP1B by defining protein binding partners for this germline-encoded metastasis modifier. Tandem affinity purification demonstrated that RRP1B physically interacts with many nucleosome binding factors, all of which have been implicated in regulation of gene expression. Examples include the linker histone H1X, poly(ADP-ribose) polymerase 1, tripartite motif-containing 28 (TIF-1β/KAP1), and cold shock domain protein A. These interactions were confirmed by co-immunofluorescence (Co-IF) and co-immunoprecipitation (Co-IP). Additionally, co-IF experiments demonstrated that RRP1B is localized to the perinuclear and perinucleolar regions, which is a particularly interesting given that factors localized to these regions are often involved in heterochromatinization and gene silencing. Furthermore, co-IP and co-IF demonstrated that RRP1B interacts with heterochromatin protein-1α and acetyl-histone H4 lysine 5, suggesting that RRP1B may well regulate gene expression through interactions with heterochromatin and euchromatin. Finally, we investigated the effects of ectopic expression of the 436Pro and 436Leu RRP1B allelic variants that have previously been associated with differential survival in breast cancer. Gene expression analyses demonstrate that, compared with ectopic expression of wildtype 436 Pro RRP1B in HeLa cells, the variant 436Leu RRP1B differentially modulates various transcription factors controlled by TRIM28 and CSDA. These data suggest that RRP1B, a tumor progression and metastasis susceptibility candidate gene, is potentially a dynamic modulator of transcription and chromatin structure. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2372.