Murine Mammary Tumor Cells Research Articles

Abstract 3862: Mechanistic studies of the metastasis suppressor prostaglandin E2 receptor EP1 in breast cancer.

Abstract Prostaglandin E2 (PGE2), the chief cyclooxygenase-2 enzyme (COX-2) product in tumors, is the predominant protumorigenic prostanoid and mediates biological effects by binding to each of four EP receptors (EP1-4). Each receptor is coupled to different intracellular signaling pathways with EP1 coupled to calcium mobilization and PKC. EP4, expressed on malignant breast cells, promotes metastasis, but a role for EP1 in metastasis has not been investigated. Our published studies indicate that EP1 was detected in the cytoplasm and nucleus of benign ducts and malignant cells in invasive ductal carcinomas, and overall survival for women with tumors negative for nuclear EP1 was significantly worse than for women with EP1 expression. Pharmacologic antagonism and reduction of EP1 expression increased metastatic capacity in our murine model of metastatic breast cancer. These data support our hypothesis that EP1 functions as a metastasis suppressor. We now report that murine metastatic mammary tumor cell lines 410.4 and 66.1 have decreased EP1 mRNA expression compared to the non-metastatic cell line 410. We have also identified the presence of a variant EP1 (EP1v) transcript. EP1v has been identified previously in murine mast cell line MC/9 and rat uterus but not in malignant cells. EP1v has a pattern of mRNA expression different than full-length EP1. Compared to the cell line 410, EP1v expression is slightly increased in 410.4 and decreased in 66.1 cell lines. In order to determine the underlying mechanism in which EP1 suppresses metastasis, a metastasis gene array was performed comparing gene expression in EP1-vector, and EP1-silenced 66.1 cell lines and several candidate genes were identified including Fn1, whose protein is altered during epithelial-to-mesenchymal transition. qPCR analysis of Fn1 expression in EP1-silenced and overexpression cell lines revealed an inverse relationship between EP1 and Fn1 expression. A decrease in EP1 expression led to a 4-5 fold increase in Fn1 expression; whereas, increased EP1 expression resulted in a 0.50 fold decrease in Fn1 expression. Like full length EP1, overexpression of EP1v also resulted in a decrease in Fn1 expression. Bioinformatic analysis of the EP1 gene identified several CpG islands. DNA methylation analysis revealed hypermethylation of the CpG island nearest to the promoter in normal, non-metastatic and metastatic murine mammary tumor cell lines. In the quest to identify a clinically relevant strategy to increase expression of this protective receptor, we treated 410.4 and 66.1 cells with demethylating agent 5-azacytidine which resulted in an increase in EP1 expression. Our published studies show that EP1 acts to suppress metastasis and we are currently exploring the contribution of EP1v to this mechanism. These findings suggest that EP1 has the potential to be a new therapeutic target in reducing breast cancer metastasis and increasing overall cancer survival. Citation Format: Jocelyn C. Reader, Xinrong Ma, Namita Kundu, Olga Goloubeva, Amy Fulton. Mechanistic studies of the metastasis suppressor prostaglandin E2 receptor EP1 in breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3862. doi:10.1158/1538-7445.AM2013-3862

Abstract 1540: TNFRs shed by tumor cells inhibit the migration of macrophages.

Abstract Breast tumor cells transform their microenvironment through the expression of pro-tumor molecules critical to their progression and metastasis. Among the targets of tumor cell secretions are macrophages, which play an essential role in tumor progression. Although macrophages possess tumor cell killing abilities, the tumor-associated macrophages (TAMs) display primarily pro-tumor functions including increasing tumor angiogenesis and promoting tumor cell invasion. Breast tumor cells shed multiple membrane-bound proteins including their tumor necrosis factor (TNF) receptors through TACE/ADAM17 activities. The release of sTNFR1/2 serves a decoy sequestering TNF and preventing TNF-driven apoptosis of tumor cells. Additionally, tumor derived sTNFR1/2 also modulate TNF effects on macrophages including their migration. Here, we investigated whether sTNFRs were shed by cells present within the breast tumor microenvironment and determined their effects on macrophage migration in response to TNF. TNF and sTNFRs concentrations were measured in murine mammary tumor and normal cells including monocytes/macrophages. In addition, the migration of murine monocytes toward TNF in the presence tumor derived soluble factors (TDSFs) was determined by transwell assays. The effects of TDSFs obtained with and without the sheddase inhibitor TAPI-0 treatment on monocyte/macrophage migration were also assessed. No TNF was detected in the TDSFs of tumor cells or secretions from normal epithelial cells. In contrast, stroma cells especially macrophages expressed high levels of TNF (p<0.001). sTNFR2 concentrations were variable in TDSFs and significantly decreased in TDSFs obtained following TAPI-0 treatment (p<0.05). Macrophages migrated toward TNF in a dose-dependent manner (p<0.05). While the addition of TDSFs prevented the macrophage migration toward TNF, the use of TDSFs collected following TAPI-0 treatment had no effects of the macrophage migration toward TNF (p<0.05). These results support the modulatory role of sTNFRs secreted by tumor cells in macrophage responses to TNF. [This works is supported by grants from the Department of Defense Era of Hope research program (BC044778) and the National Science Foundation EFRI program (CBE0736007)]. Citation Format: Stephen Rego, Rachel Helms, Alexander De Piante, Amritha Kidiyoor, Amanda Lance, Pinku Mukherjee, Didier Dréau. TNFRs shed by tumor cells inhibit the migration of macrophages. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1540. doi:10.1158/1538-7445.AM2013-1540

Abstract 3311: In vivo selection of S. typhimurium A1-R enhanced tumor-targeting variants.

Abstract The S. typhimurium A1-R mutant (A1-R), which is auxotrophic for leu-arg, and selected for high antitumor virulence, has been previously shown to be effective as monotherapy against human breast, prostate, pancreatic, and fibrosarcoma tumors that have been orthotopically implanted in nude mice. In the present study, we generated an enhanced tumor-targeting S. typhimurium variant using in vivo selection. Dual-color murine mammary tumor (MMT) cells expressing green fluorescent protein (GFP) in the nucleus and red fluorescent protein (RFP) in the cytoplasm were injected (1 × 106 cells) in both flanks of 4-week-old female nude mice. Seven days after tumor implantation, A1-R bacteria were injected (1 × 107 cfu iv). Five days after injection, the tumors were harvested and minced. The bacteria which invaded the tumor were isolated and seeded on LB agar. After overnight incubation, colonies were picked and purified and termed A1-R1. The procedure was repeated with A1-R1 to obtain A1-R2. To determine the efficacy of the new bacterial variants, MMT-dual cells were seeded onto the 6-well plates. After 48 hours incubation, A1-R, A1-R1, and A1-R2 (1 × 108 cfu/well) were added to each plate. After 0, 24, 48, 72, 96 hours incubation, each plate was observed by confocal microscopy. Compared with A1-R, the infection rate of cancer cells by A1-R1 and A1-R2 was increased. The A1-R1 and A1-R2 variant bacteria generated in vivo selection show promise for improved bacterial targeting in vivo. Citation Format: Fuminori Uehara, Yong Zhang, Yasunori Tome, Hiroki Maehara, Fuminori Kanaya, Robert M. Hoffman, Ming Zhao. In vivo selection of S. typhimurium A1-R enhanced tumor-targeting variants. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3311. doi:10.1158/1538-7445.AM2013-3311

Abstract 1488: Metastatic analysis of clonal epithelial and mesenchymal-like murine mammary tumor cells.

Abstract Tumor metastasis is the most common cause of cancer mortality, and yet it is among the most poorly understood processes in tumor progression. While carcinomas are composed primarily of epithelial cells, tumors contain a continuum of cells with epithelial and mesenchymal-like properties. This tumor cell heterogeneity is attributed to differentiation plasticity, which is characterized by epithelial to mesenchymal (EMT) or mesenchymal to epithelial transitions (MET). It has been proposed that tumor cells undergo EMT in order to disseminate from the primary tumor and MET in order to seed at secondary sites. The invasive mesenchymal carcinoma cell phenotype is thought to be a state required for tumor dissemination, but to date, pathologists have not found conclusive evidence of mesenchymal cells in human metastatic tumor samples. An alternative theory suggests that both mesenchymal and epithelial cells are required for metastasis to occur. According to this theory, detachment from the primary tumor and invasion by mesenchymal cells facilitates the movement of epithelial tumor cells that are able to seed at distant sites. We hypothesize that the presence of mesenchymal-like mammary tumor cells facilitate the metastasis of epithelial tumor cells and that only epithelial cells are able to seed at distant sites. To test this hypothesis, we cloned epithelial and mesenchymal-like primary cell lines from spontaneous mammary tumors produced in FVB MMTV/Neu mice. Epithelial cells have been stably transfected to express firefly luciferase and GFP, and we are in the process of cloning mesenchymal-like cells expressing Renilla luciferase and mCherry. We have preliminary data that shows that when clonal epithelial cells are administered into the mammary fat pad of FVB MMTV/Neu mice, tumors grow, but do not form metastatic lesions. In contrast, when stably transfected clonal epithelial cells are administered with non-transfected clonal mesenchymal-like tumor cells, luciferase+ metastatic lesions in the spleen, liver and lung are detected biophotonically, and GFP + cells are detected in the spleen and liver. These data indicate that epithelial cells are present (but does not rule out the presence of mesenchymal cells) at the metastatic site. Whether epithelial cells undergo EMT, and how metastasis is facilitated by the presence of mesenchymal cells remains to be determined. Citation Format: Katie A. Palen, Bryon D. Johnson, Jill A. Gershan. Metastatic analysis of clonal epithelial and mesenchymal-like murine mammary tumor cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1488. doi:10.1158/1538-7445.AM2013-1488

Abstract 508: Development of a human model to study the role of Glypican-3 (GPC3) on tumor progression of the mammary gland.

Abstract GPC3 was proposed as a negative regulator of cell proliferation in the mammary gland. Whereas normal breast tissues present high GPC3 levels, its expression is reduced in breast tumors. We have transfected LM3 murine mammary tumor cells with the GPC3 gene. We found that GPC3 reexpression inhibited metastasis in vivo. Although GPC3 signaling pathway is unclear, we reported that this glypican modulates Wnt, p38 and Akt pathways. However, regulation hierarchy has not been determined and we have no data in human cells. We believe it possible to generate an anti-metastasis therapy targeting GPC3 pathway. To test it, we developed a pre-clinical human model. First, we showed by qPCR that GPC3 expression levels are inversely proportional to the metastatic ability of a panel of human breast cancer cells. The metastatic Hs578T and MDA-MB231 cells expressed less GPC3 than the poorly-metastatic ZR-75-1 and MCF7 lines. We selected MDA-MB231 and MCF7 to be modified by genetic engineering. GPC3 expression was blocked by RNA interference technology. We transfected four shRNA-GPC3 and one shRNA scramble (shNC) into MCF7. After selection and cloning, we chose clone 2 that expressed about 75% less GPC3 for further characterization. On the other hand, GPC3 was reexpressed in MDA-MB231 cells by transduction of pLV-GPC3-GFP (empty vector was used as control). GPC3 mRNA increased up to 200-fold in pLV-GPC3-GFP transduced cells. Then we evaluated cell properties in the generated models. Surprisingly, MDA-MB231-GPC3 cells showed a higher proliferation rate than controls, that became significant from 48 h of exponential growth (Population doubling time= 45 h for control vs. 24 h for GPC3 reexpressing cells). On the other side, MCF7-shGPC3 bulk and clone 2 proliferated faster than -shNC. This suggests that, in this cell line, GPC3 is inhibiting proliferation. GPC3 reexpression induced a 30% inhibition on MDA-MB231 viability when cells were serum starved for 96 h, while parental and -GFP remained 100% viable. No differences were detected among MCF7 engineered cells. So, the effect of GPC3 on cell viability would depend on the cellular context. We performed a wound healing assay to evaluate migration, determining that MDA-MB231-GPC3 cells were significantly less migrant than their controls (Wound coverage: MDA-MB231-GPC3 10% vs. parental and -GFP 90%). In sum, we have developed a human cell model to study the role of GPC3 on tumor progression. Our preliminary in vitro results reveal the importance of GPC3 in the biology of breast cancer cells. Citation Format: Lilian F. Castillo, Rocio Tascon, María Amparo Lago Huvelle1, Ancély Ferreira dos Santos, Letícia Ferreira Terra, Rosangela A. m. Wailemann, Talita C. Oliveira, Mari Sogayar, Leticia Labriola, Elisa Bal de Kier Joffe, Maria Giselle Peters. Development of a human model to study the role of Glypican-3 (GPC3) on tumor progression of the mammary gland. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 508. doi:10.1158/1538-7445.AM2013-508

Abstract 555: Proliferation, apoptosis and senescence differential modulation by combining the retinoid ATRA and Protein Kinase C pharmacological inhibitors, in a bicellular murine mammary tumor cell line.

Abstract Retinoids exert some of their effects on cell differentiation and malignant phenotype reversion through interactions with specific Protein Kinase C (PKC) isoforms. In this work we studied: A)- the modulation of PKCα and PKCδ expression when cells were treated with ATRA (1 μM), B)- The expression profile of Retinoic Acid Receptors (RAR) when cells are treated with ATRA and/or PKCα / PKC δ inhibitors (Gö6976 2.5 μM / Rottlerin 1 μM) and C)- the effect of combining ATRA and PKC inhibitors on in vitro cell growth, cell cycle distribution and senescence. We used as model the murine mammary tumor-derived cell line LM38-LP, composed by luminal (LEP) and myoepithelial (MEP) cells. By RT-PCR we could determine that 48 h ATRA treatment increased PKCδ only in the LM38-LP LEP population and decreased PKCα expression levels in both cellular components. Regarding RARs profile, the treatment with PKCα inhibitor Gö6976 reduced RARγ2 levels, while its combination with ATRA also increased RARβ2. Furthermore, 96 h treatment with ATRA, Gö6976, or Rottlerin or their combinations induced cell growth inhibition (cell number x105: control 22± 1.1; ATRA 11±0.1; Gö6976 3.6±0.6; Rottlerin 9±0.1; ATRA/Gö6976 2.3±0.03; ATRA/Rottlerin 6.7±1.3, p ≤0.05 vs control in all treatments). By flow cytometry we could determine that both ATRA and Rottlerin treatments induce LM38-LP cell cycle arrest in G1 phase, while Gö6976 increased the number of cells in the subG1 phase, correlating with increased apoptosis and confirmed by Annexin V-FITC staining. Employing β-galactosidase activity assay and bright field microscopy, we determined that 96 h ATRA treatment induced senescence mainly in the MEP component while Gö6976 does it in the LEP population. Interestingly, Rottlerin treated cells showed morphological alterations compatible with autophagy. We conclude that cell growth inhibition exerted by retinoids may be enhanced by the reduction in PKCα expression and / or activity. In addition the combination of ATRA plus Gö6976, induced differential responses on the different LM38-LP cell components, showing senescence in MEP cells, together with cell cycle arrest and apoptosis mainly in the LEP compartment. Consistently, all these biological effects correlated with an increase in RARβ2 (known to induce cell differentiation) and a decrease in RARγ2 (implicated in proliferation) mRNAs level. Citation Format: Damian E. Berardi, Maria I. Diaz Bessone, Carolina Flumian, Stefano M. Cirigliano, Elisa D. Bal de Kier Joffe, Alejandro J. Urtreger, Laura B. Todaro. Proliferation, apoptosis and senescence differential modulation by combining the retinoid ATRA and Protein Kinase C pharmacological inhibitors, in a bicellular murine mammary tumor cell line. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 555. doi:10.1158/1538-7445.AM2013-555

Expression of membrane anchored cytokines and B7-1 alters tumor microenvironment and induces protective antitumor immunity in a murine breast cancer model

Many studies have shown that the systemic administration of cytokines or vaccination with cytokine-secreting tumors augments an antitumor immune response that can result in eradication of tumors. However, these approaches are hampered by the risk of systemic toxicity induced by soluble cytokines. In this study, we have evaluated the efficacy of 4TO7, a highly tumorigenic murine mammary tumor cell line, expressing glycosyl phosphatidylinositol (GPI)-anchored form of cytokine molecules alone or in combination with the costimulatory molecule B7-1 as a model for potential cell or membrane-based breast cancer vaccines. We observed that the GPI-anchored cytokines expressed on the surface of tumor cells greatly reduced the overall tumorigenicity of the 4TO7 tumor cells following direct live cell challenge as evidenced by transient tumor growth and complete regression within 30 days post challenge. Tumors co-expressing B7-1 and GPI-IL-12 grew the least and for the shortest duration, suggesting that this combination of immunostimulatory molecules is most potent. Protective immune responses were also observed following secondary tumor challenge. Further, the 4TO7-B7-1/GPI-IL-2 and 4TO7-B7-1/GPI-IL-12 transfectants were capable of inducing regression of a wild-type tumor growing at a distant site in a concomitant tumor challenge model, suggesting the tumor immunity elicited by the transfectants can act systemically and inhibit the tumor growth at a distant site. Additionally, when used as irradiated whole cell vaccines, 4TO7-B7-1/GPI-IL-12 led to a significant inhibition in tumor growth of day 7 established tumors. Lastly, we observed a significant decrease in the prevalence of myeloid-derived suppressor cells and regulatory T-cells in the tumor microenvironment on day 7 post challenge with 4TO7-B7-1/GPI-IL-12 cells, which provides mechanistic insight into antitumor efficacy of the tumor-cell membrane expressed IL-12. These studies have implications in designing membrane-based therapeutic vaccines with GPI-anchored cytokines for breast cancer.

Anti‐tumor effect of SLPI on mammary but not colon tumor growth

Secretory leukocyte protease inhibitor (SLPI) is a serine protease inhibitor that was related to cancer development and metastasis dissemination on several types of tumors. However, it is not known the effect of SLPI on mammary and colon tumors. The aim of this study was to examine the effect of SLPI on mammary and colon tumor growth. The effect of SLPI was tested on in vitro cell apoptosis and in vivo tumor growth experiments. SLPI over-expressing human and murine mammary and colon tumor cells were generated by gene transfection. The administration of murine mammary tumor cells over-expressing high levels of SLPI did not develop tumors in mice. On the contrary, the administration of murine colon tumor cells over-expressing SLPI, developed faster tumors than control cells. Intratumoral, but not intraperitoneal administration of SLPI, delayed the growth of tumors and increased the survival of mammary but not colon tumor bearing mice. In vitro culture of mammary tumor cell lines treated with SLPI, and SLPI producer clones were more prone to apoptosis than control cells, mainly under serum deprivation culture conditions. Herein we demonstrated that SLPI induces the apoptosis of mammary tumor cells in vitro and decreases the mammary but not colon tumor growth in vivo. Therefore, SLPI may be a new potential therapeutic tool for certain tumors, such as mammary tumors.

Abstract 327: Properties associated with cancer progression in primary clonal epithelial and mesenchymal-like murine mammary tumor cells

Abstract The majority of cancer patients die from tumor relapse. Consequently, characteristics of tumor cells that may contribute to aggressive cancer progression are under intense investigation. Tumors are composed of heterogeneous populations of cells that express a continuum of epithelial and mesenchymal markers. There is mounting evidence that mesenchymal tumor cells acquire invasive features and are refractory to conventional treatment, but in order to decipher the contribution of both epithelial and mesenchymal-like tumor cells to disease relapse we must understand the cellular and molecular properties of each cell type. Using a combination of different culture conditions and cell sorting strategies, we cloned epithelial and mesenchymal-like tumor cells that were derived from FVB Tg MMTV/Neu spontaneous mammary tumors. The availability of these clonal primary cell lines provides an unique opportunity to compare tumor cell properties that may contribute to aggressive disease such as proliferation, resistance to apoptosis, tumorigenicity, invasiveness, expression of growth factor and cytokine receptors, inflammatory cytokine secretion, immunogenicity, and expression or down-regulation of immune-modulatory molecules. Some of our preliminary data has revealed that (1) Epithelial cells are more proliferative in vitro, and cell proliferation was reduced upon exposure to the tyrosine kinase inhibitor, TKI-258. TKI-258 did not significantly decrease the proliferation of mesenchymal-like tumor cells, (2) A population of epithelial-enriched cells was more tumorigenic as compared to mesenchymal-like-enriched cells when administered to an immune competent syngeneic host, (3) Epithelial cells express greater Neu transgene, IL-6 and TNF-α transcripts than mesenchymal-like cells, and (4) When low passage clonal epithelial and mesenchymal-like cells were used as a whole cell tumor vaccine in FVB wild-type mice there was an increase in CD8 T cell activation as compared to that elicited by administration of a non-clonal high passage tumor cell line. We are continuing to explore molecular and functional differences between the clonal epithelial, clonal mesenchymal and the non-clonal tumor cell line cells. Characterization of epithelial and mesenchymal-like tumor cells represents an initial step in deciphering how tumor cells contribute to aggressive cancer progression and will offer insight into how epithelial and mesenchymal-like tumor cells respond to therapeutic interventions. The ultimate goal will be to tailor anticancer therapy toward eradicating the unique type of tumor cells that populate patient tumors. 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 327. doi:1538-7445.AM2012-327

Abstract 1408: Activation status and function of the canonical Wnt pathway in murine mammary tumor cells reexpressing Glypican-3 (GPC3)

Abstract GPC3 is a proteoglycan downregulated in breast cancer cells. We previously demonstrated that GPC3 reexpression inhibits in vivo metastatic potential of LM3 murine mammary adenocarcinoma cells. This event was associated in vitro with a lower motility and an increased aggregation, as well as with an enhanced senescence and apoptosis. We have previous evidence indicating that the canonical Wnt pathway is inhibited in LM3-GPC3 cells. The aim of this work was to validate the inhibition of canonical Wnt signaling in GPC3 reexpressing cells and to investigate whether differences in cell behavior are due to the regulation of GPC3 on this pathway. By means of western blots (WB) we established that LM3-GPC3 cells have 5-10 folds less phosphorylated GSK3B. Using cytoplasmic protein extracts and WB we also determined that GPC3 reexpressing cells present lower levels of cytoplasmic B-Catenin. This was further studied by immunofluorescence (IF), where it was found that while LM3-vector cells show positive signal for B-catenin mainly in nucleus and cytoplasm, this staining is restricted to the membrane and cytoplasm in LM3-GPC3 cells. In addition, a gene reporter assay employing OT/OF plasmids confirmed the canonical Wnt pathway inhibition induced by GPC3. To determine whether cell properties modulated by GPC3 are mediated by the canonical Wnt pathway, we treated cells with its activator LiCl (or NaCl as control). LiCl treatment increases 10-fold the phosphorylation of GSK3B, as compared to LM3-GPC3 cells treated with NaCl, and induces an enhance of about 50% of the canonical Wnt transcriptional activity (gene reporter assay). We confirm that although LiCl partially reverted senescence induced by GPC3 reexpression (Senescent X-Gal positive cells: LM3-GPC3+NaCl= 85% vs. LM3-GPC3+LiCl= 65%, p<0.05), it had no effects on apoptosis. We established by wound healing assays that LM3-GPC3 cells +LiCl recover their ability to migrate (Coverage of the wound: LM3-GPC3+NaCl= 15% vs LM3-GPC3+LiCl= 75%, p<0.05). Finally, LiCl treatment reduced LM3-GPC3 homotypic cell aggregation, reaching similar levels to LM3-vector clones (p<0.05). In summary, we confirm by a new methodological approach that GPC3 inhibits the canonical Wnt signaling. Our results suggest that GPC3 would be modulating senescence, migration and cell aggregation of mammary tumor cells through this pathway inhibition. 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 1408. doi:1538-7445.AM2012-1408

Abstract 5328: Interleukin-4 receptor regulates metastasis of mammary epithelial cancer cells

Abstract Interleukin-4 (IL-4), a cytokine produced mainly by immune cells, may promote cancer cell survival by mediating apoptosis resistance in epithelial tumors. The IL-4 Receptor (IL-4R) is overexpressed in a variety of human solid cancer cell lines and primary cell lines, and may be a promising target for anti-tumor therapy. Here, we show that loss of IL-4Rα, a component of the IL-4 receptor, in two murine mammary tumor cell lines (R221A and 4T1), reduces tumor cell survival and results in attenuated metastatic growth in secondary sites. Proliferation and direct cell counting analyses, following knockdown of IL-4Rα with RNA interference, show that R221A clones with reduced IL-4Rα expression proliferate at a comparable rate to control clones, but the number of cells is significantly reduced. This data correlates with the finding that reduced IL-4Rα expression in R221A cells almost completely abolishes tumor take after orthotopic mammary injection in vivo. Furthermore, reduced IL-4Rα expression in R221A and 4T1 cell lines results in a decrease [2.4 fold] in lung metastatic growth in vivo. Reduced expression of IL-4Rα in 4T1 cells results in a similar decrease of liver metastasis. In support of this data, proliferation assays following serum starvation and a modified clonogenic survival assay show that both 4T1 and R221A knockdown clones have a decrease [2 fold] in survival ability in comparison to control clones. Taken together, our results indicate that the IL-4 receptor promotes survival of breast cancer cells at metastatic sites, and suggests that drugs targeting this pathway, currently in clinical trials for respiratory ailments, may have potential for limiting metastatic disease. 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 5328. doi:1538-7445.AM2012-5328

Abstract 448: Expression of Glypican-3 (GPC3) in breast cancer tumors from Brazilian and Argentinean patients

Abstract The heparan sulfate proteoglycan Glypican-3 (GPC3) is involved in the signaling regulation of several growth factors. GPC3 is widely expressed in embryonic tissues, but disappears from most adult tissues, except for the mammary gland, among others. Several studies have involved GPC3 with cancer and some data indicate that this glypican is diminished in breast tumors. Previously, we have transfected the murine mammary tumor cell line LM3 (GPC3 negative) with the GPC3 complete cDNA sequence. We found that GPC3 reexpression is able to inhibit invasion and metastasis in vivo as well as to induce an in vitro EMT reversion, suggesting that GPC3 could act as a metastasis suppressor. The aim of the present work was to determine, in a prospective trial, whether GPC3 expression might be useful as a biomarker able to predict metastasis outcome in breast cancer patients. Our local access to a major source of breast human tissues from a representative Latin American population is an exceptional tool for the development of this new biomarker. A preliminary study was perform to analyze the expression of GPC3 at the mRNA level, by quantitative real time RT-PCR, in breast cancer samples (Brazilian n=15, Argentinean n=22) and peri-tumoral normal breast tissues (Brazilian n=16, Argentinean n=4). Even though the analyzed sample size was small, we found that both Brazilian and Argentinean tumor populations presented similar characteristics related to GPC3 expression. It was established a lower GPC3 level in tumor (n=37) than in peri-tumoral normal tissues (n=20) (p<0.001, Wilcoxon test). In matched samples, we found that 7 out of 8 tumors expressed less GPC3 than their corresponding normal peri-tumoral tissue. On the other hand, we studied by univariate analysis, the association of GPC3 expression with the parameters frequently used in the management of breast cancer patients, such as age, status, tumor size and stage, histology, metastatic axillary nodules, histological grade, nuclear grade, mitotic index, hormonal receptors, as well as with the disease-free survival probability (DFS). No association was found between GPC3 expression and these clinical-pathological parameters (Chi square and Pearson correlation tests). These results suggest the independence of GPC3 expression. The Kaplan-Meier curves and the Log Rank test indicated that GPC3 expression was not able to predict the DFS in our population of breast cancer patients. However, interestingly the only two patients who relapsed were those that expressed very low relative levels of GPC3. We believe that a longer patients’ follow-up, as well as a higher number of samples, will be needed in this prospective study to determine the usefulness of GPC3 as a biomarker to predict metastasis. 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 448. doi:1538-7445.AM2012-448

Abstract 2231: The over-activation of Akt1 oncogene heightens the Glypican-3 (GPC3) effects on metastatic progression of murine mammary tumor cells

Abstract It is known that Akt kinases 1, 2 and 3 have critical roles in regulating survival, metabolism and other cellular activities. However, their functions on migratory potential and metastasis development are less clear. GPC3 is a proteoglycan downregulated in breast tumors. We have previously shown that GPC3 reexpression inhibits metastatic capacity in vivo of the LM3 murine mammary adenocarcinoma cells. Moreover, this phenomenon was associated with a partial reversion of the epithelial-to-mesenchymal transition as shown in vitro. On the other hand, we have also determined that GPC3 modulates Akt signaling pathway in LM3 cells. The aim of this work was to investigate the role of Akt1 on the invasive/metastatic behavior of GPC3 reexpressing cells. First, we have confirmed that LM3-GPC3 cells present higher Akt1 levels than LM3-vector ones by western blot. To elucidate the function of this isoform, LM3-GPC3 clones were infected with viruses carrying a constitutively active variant of Akt1 (CA Akt1). Although there were no changes in the actin cytoskeleton organization, we could determine by a wound healing assay that LM3-GPC3-CA Akt1 cells were less migrant than their controls (Wound coverage: LM3-GPC3 control=50% vs. LM3-GPC3-CA Akt1=30%, p<0.05). To address an in vivo effect, different clones were injected s.c. into syngeneic female BALB/c mice. We found that these tumors did not differ in their growth rate, reaching a similar size at 30 days post inoculation (LM3-vector= 189±25, LM3-GPC3= 181±40, LM3-GPC3-CA Akt1= 200±51 mm3). No differences were found in either tumor incidence or in tumor latency. In order to study the experimental metastatic ability, all cell lines were i.v. inoculated. We observed that LM3-GPC3-CA Akt1 tumors developed fewer lung metastases than controls (Median [Range]: LM3-GPC3 control=9 [3-29] vs. LM3-GPC3 CA Akt1=0.5 [0-10], p<0.05). Moreover, the LM3-GPC3-CA Akt1 metastatic nodes were also of a smaller size (<0.5 mm). In sum, our results suggest that Akt1 expression and signaling could be mediating GPC3 inhibitory effect on the metastatic dissemination. The over-activation of this oncogene would exacerbate or heighten GPC3 effects, preventing the metastasis progression through the reduction of cell migration. 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 2231. doi:1538-7445.AM2012-2231

Abstract 3015: Crosstalk between ≤ and ≤ PKC isoforms and retinoic acid system in malignant phenotype reversion

Abstract Retinoids may exert some of their effects on cell differentiation and malignant phenotype reversion through interaction with different PKC isoforms. In this work we studied the crosstalk between retinoid acid system (RA) and PKC signaling pathway and its implication in malignant phenotype reversion using a murine mammary tumor cell line (LM3) and a human breast cancer-derived cell line (MDA-MB231). ATRA (all trans RA) treatment (1uM,72h) induced a significant growth inhibition in vitro in LM3 cell line. This result was confirmed in vivo when LM3 cells were injected orthotopically in female BALB/c mice while carrying subcutaneous slow release ATRA pellets (10 mg). This phenomenon was associated with the reduction of pErk1 levels (80±9%) and the increase of the cell cycle inhibitor p27 in the nuclear fraction without altering Cyclin D1 expression in LM3 cell line. None of these modulations were observed in ATRA unresponsive MDA-MB231 cell line. We determined by Western blot that 24h treatment with ATRA induced an increase of PKC∈ and PKC∈ levels, detecting alpha isoform only in the membrane fraction and delta isoform in the nuclear fraction. In contrast, in MDA-MB231 cells, PKC∈ and PKC∈ expression was reduced after ATRA exposure. Interestingly, pharmacological inhibition of PKC∈ and PKC∈ prevented retinoid receptors activation by ATRA in LM3 cells, as evidenced by a reporter gene assay (RARE-Luciferase). Western blot showed that pharmacological inhibition of PKC∈ (Rottlerin 1μM, 24h) impaired ATRA-induced RARα1 translocation to the nucleus while the pharmacological inhibition of PKC∈ (Gö6976 5μM, 24h) did not affect this translocation. Moreover, immunoprecipitation assays showed that only PKC∈ co-immunoprecipitated with RARα1 after ATRA treatment, suggesting a physical interaction between both molecules. Rottlerin-ATRA treatment (72h) reversed the effect on proliferation rate exerted by retinoid treatment. Gö6976-ATRA treatment (72h) significantly decreased cell duplication rate compared with ATRA or Gö6976 treatment alone, in an additive manner. Pre-treated LM3 cells with Gö6976 were intravenously injected in female BALB/c mice that carried subcutaneous slow release ATRA pellets in order to evaluate the importance of combination therapy in the last stages of metastatic spread. We noticed that this combined therapy produced a higher decrease in the number of lung metastases, suggesting an additive effect. Our results indicate that PKC/Retinoid Crosstalk is implicated in growth inhibition through the induction of RA transcription genes that lead to cell cycle arrest and differentiation. Finally we speculate that retinoid treatment combined with PKC∈ isoform modulators could be considered for treatment of PKC∈ positive breast cancer patients. 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 3015. doi:1538-7445.AM2012-3015

Abstract 2845: Prostaglandin E receptor EP4 antagonist RQ00015986 inhibits breast cancer metastasis and protects natural killer cells from PGE-mediated immune suppression

Abstract The cyclooxygenase-2 (COX-2) pathway is upregulated in breast and other malignancies. The COX-2 product prostaglandin E2 (PGE2) promotes tumor growth and metastasis by acting on a family of G-protein-coupled receptors (EP1-EP4). In breast and colon malignancies, EP4 is a principle mediator of pro-tumorigenic and pro-metastatic activities. Our previous studies demonstrated that targeting EP4 with shRNA or by pharmacologic antagonists of EP4 leads to reduced metastasis in a preclinical model of metastatic breast cancer. These studies and others establish EP4 as a potential therapeutic target, however, there is a paucity of effective EP4 antagonists available for preclinical and clinical investigation. Using a novel orally available EP4 selective antagonist, RQ00015986, we have evaluated efficacy in a syngeneic murine model of metastatic breast cancer. Murine and human breast cancer cells express EP4 and receptor occupation with PGE2 (binds all EP) or with PGE1-OH (EP4 agonist) results in adenyl cyclase activation and elevated intracellular cAMP. RQ00015986, in uM/L concentrations, was able to prevent EP4-mediated adenyl cyclase activation in murine mammary tumor cell lines 66.1 and 410.4. Breast tumor cells migrate in response to either PGE2 or PGE1-OH and RQ00015986 inhibited this response. Pretreatment of 66.1 cells with RQ00015986 prior to i.v. injection into syngeneic mice resulted in 58-69% inhibition in numbers of lung tumor colonies. Natural Killer (NK) cells are critical to the control of metastatic dissemination. The ability of RQ00015986 to inhibit lung tumor colonization was completely lost in hosts depleted of mature NK cells. We have reported previously that NK cell functions in tumor-bearing mice are severely depressed through the actions of tumor-derived PGE2 acting on EP4 receptors expressed on the NK cell. The capacities of NK cells to migrate, to produce cytokines and to lyse mammary tumor target cells are each inhibited through PGE2. In the presence of RQ00015986, the capacity of cultured NK cells to migrate and to produce Interferon-α was restored to normal levels. More interestingly, treatment of tumor-bearing mice with RQ00015986 in vivo also restored NK cell functions. This report provides evidence that a novel EP4 antagonist may have therapeutic potential in cancer and may act by antagonizing EP4 prometastatic functions on the malignant cell as well as protecting NK effector cells from PGE2-mediated immune 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 2845. doi:1538-7445.AM2012-2845

Caveolin-1 expression is elevated in claudin-low mammary tumor cells

BackgroundCaveolin-1 is a scaffolding protein found in plasma membrane invaginations known as caveolae. Caveolin-1 can regulate a number of intracellular processes such as signal transduction, cholesterol metabolism and vesicular transport. With respect to breast cancer caveolin-1 has been observed in both tumor cells and stromal cells surrounding tumors however most of the recent research has focused on how the loss of caveolin-1 in the stromal cells surrounding the tumor alters the tumor microenvironment.MethodsCaveolin-1 expression was evaluated in (1) mammary tumors induced by the transgenic overexpression of the type I insulin-like growth factor receptor (IGF-IR), (2) mammary tumors that became independent of IGF-IR signalling and acquired a claudin-low genotype, (3) two murine mammary epithelial tumor cell lines and (4) two murine mammary claudin-low tumor cell lines.ResultsWe found that mammary tumors induced by IGF-IR overexpression expressed low levels of caveolin-1 while mammary tumors that became independent of IGF-IR signalling expressed considerably higher levels of caveolin-1. Interestingly, pockets of caveolin-1 positive cells could be observed in some of the IGF-IR-induced mammary tumors and these caveolin-1 positive cells were associated with tumor cells that expressed basal cytokeratins (cytokeratins 5 and 14). This caveolin-1 expression pattern was maintained in the murine mammary tumor cell lines in that the epithelial mammary tumor cell lines expressed little or no caveolin-1 while the claudin-low cell lines expressed caveolin-1.ConclusionsOur model indicates that mammary tumor cells with epithelial characteristics lack caveolin-1 while mesenchymal tumor cells express caveolin-1 suggesting that caveolin-1 may serve as a marker of mammary tumor cells with mesenchymal characteristics such as claudin-low breast tumors.

Separation and Characterization of Epithelial and Mesenchymal-like Murine Mammary Tumor Cells Reveals Epithelial Cell Differentiation Plasticity and Enhanced Tumorigenicity of Epithelial-enriched Tumor Cells

Tumors are composed of heterogeneous populations of cells including tumor-initiating cells (TICs) and metastatic precursors. While the origin of these cells is unknown, there is evidence that tumor cells can transdifferentiate from an epithelial to a mesenchymal phenotype, a property referred to as epithelial-to-mesenchymal transition (EMT). This cellular plasticity may explain the heterogeneous nature of tumors and differences in the tumorigenic and invasive properties of cells. Understanding the origin of these cells and the contribution of external factors that influence the acquisition of cellular properties is critical for the development of therapeutics to eradicate cancer. In this study, we show that primary murine tumor cells harvested from FVB/N Tg (MMTV/Neu) spontaneous mammary tumors possess differentiation plasticity and can be enriched to be epithelial or mesenchymal-like using selected culture media conditions, and we show evidence of EMT in a clonal population of primary epithelial tumor cells when cultured in fibroblast growth factor-1 (FGF-1) or transforming growth factor-β (TGF-β). We also determined that in contrast to the identification of mesenchymal-like tumor cells as TICs in orthotopic xenograph models of tumorigenicity, epithelial-enriched murine mammary tumor cells were more tumorigenic as compared to mesenchymal-enriched cells when transplanted back subcutaneously into syngeneic immune competent mice. Together, these data suggest that EMT plasticity can be induced in primary murine mammary tumor cells, and that tumorigenicity of epithelial or mesenchymal-like cells may be influenced by factors such as the site of tumor inoculation or the immune state of the host (xenogenic immune compromised versus syngeneic immune competent).

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.

S6-5: Obesity Drives Epithelial-to-Mesenchymal Transition and Tumor Progression in a Novel Claudin-Low Mammary Cancer Model.

Abstract Background: Epidemiological evidence suggests a potential role of obesity in regulating clinical subtype, differentiation status, and prognosis of breast cancer. Specifically, an elevated waist-hip ratio is associated with increased risk and progression of basal-like breast cancer, an aggressive form characterized by heterogeneous tumors typically enriched in a putative tumor initiating cell (TIC) population. However, the exact mechanism of obesity-driven tumor progression remains unknown. Therefore, we hypothesized that obesity regulates a plastic population of multipotent malignant cells. Materials, Methods, and Results: To test this hypothesis, we generated and characterized two distinct murine mammary tumor cell lines derived from MMTV-Wnt-1 transgenic mice, designated M-Wnt and E-Wnt. M-Wnt cells displayed a mesenchymal morphology while E-Wnt cells had an epithelial morphology. M-Wnt cells harbored a large CD44+/CD24- putative TIC population (62% +/−7.8), had significant mammosphere forming capacity (>30% of cells form mammospheres, p<0.0001), and increased ALDH activity (7% are ALDH+, p=0.0004). M-Wnt cells have increased migration (scratch assay) and invasion in vitro (226-fold higher after 30h, p<0.0001). As few as 50 unsorted M-Wnt cells, injected into C57BL/6 mice were capable of forming a tumor. Microarray analysis revealed that M-Wnt cells display gene expression profiles virtually identical to human Claudin-low breast tumors, while E-Wnt cells clustered with basal-like tumors. EMT and stem cell gene expression patterns of M-Wnt cells were maintained in vivo, including decreased E-cadherin and increased N-cadherin, fibronectin, vimentin, SNAIL, TWIST, SLUG, FOXC2, and TGF-β (p<0.05 for all). Using these cell lines in vivo, we tested the hypothesis that energy balance modulation, through diet-induced obesity (DIO) and calorie restriction (CR), regulates the TIC population. We found that M-Wnt tumors, transplanted into ovariecomized syngeneic female C57BL/6 mice, grew at significantly different growth rates depending on the diet treatment (DIO > Control, p=0.011; CR < Control, p=0.012), while E-Wnt tumors were only affected by CR (CR < Control p=0.001; no difference between DIO and Control tumors). DIO enhanced M-Wnt tumor progression, adipocyte infiltration, and central necrosis and drove EMT (decreased E-Cadherin and increased fibronectin and N-Cadherin expression) through modulation of key TIC associated genes, including increased TGF-β, SNAIL, FOXC2, and Oct4 (p<0.05 for all). Discussion: In conclusion, we found that the mesenchymal M-Wnt cell line is highly responsive to changes in dietary energy balance status relative to the E-Wnt differentiated epithelial cell line, which demonstrates that clinical subtypes of breast cancer are differentially regulated by energy balance modulation. Additionally, our data demonstrates, for the first time, that energy balance modulation (ie, CR and obesity) directly regulates EMT, in response to local upregulation of TGF-β signaling. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr S6-5.

Anti-Transforming Growth Factor ß Antibody Treatment Rescues Bone Loss and Prevents Breast Cancer Metastasis to Bone

Breast cancer often metastasizes to bone causing osteolytic bone resorption which releases active TGFβ. Because TGFβ favors progression of breast cancer metastasis to bone, we hypothesized that treatment using anti-TGFβ antibody may reduce tumor burden and rescue tumor-associated bone loss in metastatic breast cancer. In this study we have tested the efficacy of an anti-TGFβ antibody 1D11 preventing breast cancer bone metastasis. We have used two preclinical breast cancer bone metastasis models, in which either human breast cancer cells or murine mammary tumor cells were injected in host mice via left cardiac ventricle. Using several in vivo, in vitro and ex vivo assays, we have demonstrated that anti-TGFβ antibody treatment have significantly reduced tumor burden in the bone along with a statistically significant threefold reduction in osteolytic lesion number and tenfold reduction in osteolytic lesion area. A decrease in osteoclast numbers (p = 0.027) in vivo and osteoclastogenesis ex vivo were also observed. Most importantly, in tumor-bearing mice, anti-TGFβ treatment resulted in a twofold increase in bone volume (p<0.01). In addition, treatment with anti-TGFβ antibody increased the mineral-to-collagen ratio in vivo, a reflection of improved tissue level properties. Moreover, anti-TGFβ antibody directly increased mineralized matrix formation in calverial osteoblast (p = 0.005), suggesting a direct beneficial role of anti-TGFβ antibody treatment on osteoblasts. Data presented here demonstrate that anti-TGFβ treatment may offer a novel therapeutic option for tumor-induced bone disease and has the dual potential for simultaneously decreasing tumor burden and rescue bone loss in breast cancer to bone metastases. This approach of intervention has the potential to reduce skeletal related events (SREs) in breast cancer survivors.