Murine Mammary Tumor Cell Line 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 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.

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 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.

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.

Abstract 546: Effects of high-dose radiotherapy on the bone marrow microenvironment in the 4T1 model of breast cancer

Abstract Background: Of patients with breast cancer, an estimated 40 percent relapse and develop metastatic disease with bone as a preferred site of metastases. Radiation therapy is used frequently to treat bone metastases for palliation of symptoms and to improve local control. Advances in the field of radiation oncology such as image-guided and intensity-modulated radiation therapy (IGRT/IMRT) permit the delivery of high-dose radiation to bone metastases. The tumor stroma and bone marrow-derived cells have been shown to impact the response to irradiation, potentially via radiation-induced recruitment of bone marrow-derived cells and mesenchymal stem cells. To date, little is known about changes in the bone marrow and tumor microenvironment following high-dose irradiation. Using a murine model of metastatic breast cancer, we sought to investigate the effect of local high-dose irradiation on the irradiated, non-irradiated tumor microenvironments, and on the pattern of circulating hematopoietic, endothelial progenitor and mesenchymal stem cells. Methods: The metastatic murine mammary tumor cell line, 4T1, was engineered to express GFP and luciferase for in vitro and in vivo detection of tumor cells. These cells were injected into the bilateral tibias of BALB/c mice. Five days following intra-tibial injection, animals received either high-dose irradiation or sham irradiation to the right hind limb. Growth and progression of bone and lung metastases were monitored by in vivo bioluminescence. Blood was assayed for circulating myeloid-derived suppressor cells (MDSC), endothelial progenitor cells (EPC), and mesenchymal stem cells (MSC) at several time points following irradiation. Results: Intra-tibial injection of 4T1 cells resulted in rapid tumor growth and development of pulmonary metastases. High-dose irradiation (20Gy) resulted in a significant reduction in the irradiated tumor size without a significant change in the size of contralateral non-irradiated tumor, as evident by bioluminescent imaging. Pulmonary metastases in irradiated animals were hemorrhagic as compared to non-irradiated animals. Irradiation resulted in increased macrophage recruitment to the irradiated, but not the non-irradiated tumor. As compared to control mice and irradiated non-tumor bearing mice, animals with 4T1 intra-tibial tumors have increased numbers of circulating MDSC (CD11b+/Gr1+), EPC (CD144+/VEGFR2+), and MSC (CD105+/CD29+). Conclusions: Here we introduce a model of breast cancer bone metastasis that may be utilized to study the effects of high-dose irradiation. Irradiation results in changes in immune cell composition at both the local and systemic levels. Ongoing studies will address the specific immune cell subtypes present in irradiated versus non-irradiated bone tumors and the corresponding pulmonary metastases. 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 546. doi:10.1158/1538-7445.AM2011-546

Abstract 1456: Prostaglandin E2 receptor EP1 as a metastasis suppressor in breast cancer

Abstract The cyclooxygenase-2 enzyme (COX-2) is highly expressed in breast cancer and contributes to the metastatic process. Inhibition of COX-2 can decrease breast tumor growth and metastasis. Prostaglandin E2 (PGE2), the chief COX-2 product in tumors, has been identified as a predominant protumorigenic prostanoid. PGE2 mediates biological effects by binding to each of four EP receptors (EP1-4). EP1 is coupled to calcium mobilization and PKC; whereas, EP2 and EP4 activate PKA/cAMP, PI3K and ERK pathways. EP3 generally inhibits cAMP levels. EP4, expressed on malignant breast cells, promotes metastasis, but a role for EP1 in metastasis has not been investigated. Using a combination of immunohistochemistry, pharmacologic and genetic approaches, we studied EP1 expression in human breast tissue and function in a murine model of breast cancer metastasis. Our published studies indicate that EP1 was detected by immunohistochemistry in the cytoplasm and nucleus of benign ducts and malignant cells in invasive ductal carcinomas. Overall survival for women with tumors that were negative for nuclear EP1 was significantly worse than for women with EP1 expression. Additionally, pharmacologic antagonism and reduction of EP1 expression by shRNA increased metastatic capacity in our murine model of metastatic breast cancer. These data suggested that EP1 may function as a metastasis suppressor. We now report that murine mammary tumor cell lines 410, 410.4 and 66.1 have decreased mRNA levels of EP1 compared to normal murine mammary epithelial cell line EpH4. The EP1 receptor is normally coupled to calcium mobilization. Even though EP1 expression is decreased in mammary tumor cell lines, preliminary studies suggest the existence of functional EP1 receptor as PGE2 is capable of inducing calcium mobilization in 410.4 cells. In addition, treatment of 410 and 66.1 cells with 5-azacytidine is capable of increasing EP1 mRNA levels which may identify a clinically relevant strategy to increase expression of this protective receptor. We have also identified the presence of a variant EP1 (EP1v) transcript in this murine metastatic breast cancer model. EP1v has been identified previously in murine mast cell line MC/9 and in rat uterus; however, this is the first report that EP1v has been identified in malignant cells. Like EP1, mRNA expression of EP1v is also decreased in 410, 410.4 and 66.1 cell lines compared to EpH4 cells. 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: {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 1456. doi:10.1158/1538-7445.AM2011-1456

Abstract 609: Frondoside antagonizes the prostaglandin E receptor EP4 and inhibits breast tumor metastasis

Abstract Sea cucumber has been used as a folk medicine for many conditions and recently antitumor activities have been described. Frondoside A, a triterpenoid glycoside isolated from the sea cucumber Cucumaria frondosa, as well as the related compound Frondanol A5 inhibit growth of pancreatic, colon and breast tumor cell lines in vitro and show activity in a primary prevention model of colon cancer. The cyclooxygenase 2 pathway contributes to growth and progression of many solid tumors by multiple mechanisms. The COX-2 product, prostaglandin E2 (PGE2) contributes to malignancy by acting on a family of four G-protein-coupled receptors (EP1, EP2, EP3, EP4). We have shown that both human and murine breast tumor cell lines express all EP receptors and that EP4 contributes to metastatic potential in a syngeneic murine model of metastatic breast cancer. Other laboratories have also provided evidence that EP4 promotes malignancy but a paucity of effective EP4 antagonists has slowed the development of clinically evaluable EP-targeted compounds. Based on the reported anti-inflammatory properties of Frondoside A, we asked if this compound has EP antagonist properties. We now report the novel finding that Frondoside A is a potent antagonist of EP4 and, to a lesser degree, antagonizes EP2. Frondoside A inhibits the binding of radio-labeled PGE2 to Chem-1 cells expressing EP4 or HEK cells transduced to express EP2. Using EP4-expressing MDA-MD-231 human breast cancer or murine mammary tumor cell line 66.1, we show that EP4-mediated activation of adenylate cyclase is also blocked by Frondoside A indicating both pharmacologic and functional EP4 receptor antagonism. We determined the effect of Frondoside A on metastatic potential of line 66.1 tumor cells. Treatment of 66.1 cells with non-cytotoxic concentrations of Frondoside A prior to i.v. administration of tumor cells into syngeneic Balb/cByJ female mice reduced lung colonizing capacity by 58%. These studies describe a potent metastasis inhibitory activity of Frondoside A and show, for the first time, that this compound may be a potent novel EP4/EP2 antagonist with activity in a model of metastatic breast cancer. 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 609. doi:10.1158/1538-7445.AM2011-609

Abstract 1199: Implications of protein kinase C (PKC)α and PKCΔ on murine mammary tumor growth and metastatic dissemination; effect of retinoids

Abstract The retinoid system exerts some of its effects on cell differentiation and malignant phenotype reversion through the interaction of its receptors (RAR) with different PKC isoforms. In this work, using a murine mammary tumor cell line (LM3), we studied the interaction between RARs and PKC in vitro as well as whether the overexpression of α and Δ PKC isoforms could influence the effect of retinoids on cell proliferation both in vivo and in vitro. Western blot assays showed that the pharmacological inhibition of PKCΔ (Rottlerin 5μM, 24h) impaired all trans retinoic acid (ATRA)-induced RARα1 translocation to the nucleus while the pharmacological inhibition of PKCα (Gö6976 5μM, 24h) did not affect this translocation. Moreover, immunoprecipitation assays showed that only PKCΔ co-immunoprecipitated with RARα1 after ATRA treatment, suggesting a physical interaction between both molecules. However, both PKC isoforms seemed to be necessary for RARs activation as determined by a gene reporter assay. Next, by a stable transfection procedure, we overexpressed PKCα and PKCΔ in LM3 cell line. In vitro assays showed that LM3-PKCα cells presented the highest proliferative rate and were highly responsive to ATRA treatment reducing their growth capability. These genetically modified lines were also inoculated orthotopically into the mammary glands of female BALB/c mice. Ten days later, when tumors were palpable, mice were implanted with ATRA pellets (10 mg/mouse). Tumors overexpressing PKCα presented a higher growth rate and were more metastatic than control vector-transfected LM3 cells, but showed a significant response to ATRA treatment, impairing primary tumor growth and reducing lung metastases number [Md (range) 55 (20-75) vs. 16 (0-27) in non-treated and ATRA-treated LM3-PKCα cells respectively (P<0.05 Mann-Whitney test)]. Both control and PKCΔ overexpressing tumors were unresponsive to retinoid treatment.We can conclude that, while PKCα overexpressing cells were more aggressive than control ones, they showed an important response to retinoid treatment both in vivo and in vitro, indicating that a retinoid therapy could be successful in human mammary tumors with high PKCα levels. On the other hand, while we found an in vitro clear interaction between PKCΔ and RARα1, the overexpression of PKCΔ did not to modulate their response to the retinoid therapy. 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 1199. doi:10.1158/1538-7445.AM2011-1199

Abstract 2422: Epithelial interleukin-4 receptor promotes metastasis of mammary tumor cells

Abstract Interleukin-4 (IL4) is known primarily for its immune system functions where it can stimulate lymphocytes and promote macrophage activation. Various cancer cells, including breast, are now known to express functionally active IL4 receptors, which appear to play a role in cell survival. We believe that blocking IL4 signaling specifically in epithelial cells will attenuate metastasis because of reduced cell survival in secondary sites. To test this hypothesis, we have generated multiple clones of two murine mammary tumor cell lines that show efficient knockdown of an essential component of the IL4 receptor, IL4-receptor [IL4R] alpha. Initial characterization of knockdown and control clones showed a significantly slower growth rate in vitro as assessed both by metabolic (MTT) assays and direct cell counting. Flow cytometric analysis using either annexin V staining or sub G1 measurement showed that clones lacking IL4Ralpha had 2-fold higher basal levels of apoptosis. To test effects in vivo, we have performed experimental and spontaneous metastasis assays using clones of the 4T1 cell line. Both pulmonary and hepatic metastatic lesions were significantly decreased [2.4-fold lower] in mice injected with IL4Ralpha knockdown clones compared to controls. There was however no significant effect on the primary mammary tumors. Together these results suggest that IL4 signaling in epithelial tumor cells is pro-metastatic, and blocking it may be a useful therapeutic approach for preventing metastatic spread. 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 2422. doi:10.1158/1538-7445.AM2011-2422

Abstract 3332: Obesity drives epithelial/mesenchymal transition and tumor progression in a novel Wnt-1 mammary cancer model

Abstract 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 breast cancer stem cell (BCSC) population. Therefore, we hypothesized that obesity regulates a plastic population of multipotent malignant cells. To test this hypothesis, we generated and characterized two distinct murine mammary tumor cell lines derived from MMTV-Wnt-1 transgenic mice. M-Wnt cells displayed a mesenchymal morphology while E-Wnt cells had an epithelial morphology. M-Wnt cells harbored a large CD44+/CD24- putative cancer stem cell 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 BCSC 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 BCSC associated genes, including increased TGF-β, SNAIL, FOXC2, and Oct4 (p<0.05 for all). 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, suggesting that energy balance modulation (ie, CR and obesity) regulates tumor growth through direct regulation of a BCSC population, possibly in response to alterations in TGF-β signaling. 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 3332. doi:10.1158/1538-7445.AM2011-3332

Abstract 339: Importance of retinoic acid receptors alpha, beta and gamma on the in vivo tumor growth of a murine mammary bi-cellular tumor cell line: Differential response of luminal and myoepithelial components to retinoids treatment

Abstract The incidence of breast cancer has apparently increased throughout the world during the last century. Caucasian women in the western world have a considerably higher breast cancer risk. Due to their role in the regulation of cell growth and differentiation through binding to RAR/RXR receptors, retinoids (Rds) are being evaluated in clinical trials of cancer prevention. We focus our studies in LM38-LP, a murine mammary tumor cell line composed by luminal (LEP) and myoepithelial (MEP) cells that express all functional retinoic acid receptors RAR/RXR. We studied the mechanisms involved in Rds effects in LEP/MEP interactions. By using anti E-cadherin/immunobeads we managed to temporarily separate LEP and MEP compartments in LM38-LP cell line. We determined that ATRA (all trans retinoic acid) induced a significant increase of RARb2 and RARg2 only in LEP cells (RT-PCR). Rds treatment increased the adhesive capacity of the bicellular LM38-LP cell line to fibronectin (FN). When the two components of the cell line were analyzed separately, Rds only induced an increase in the adhesion to FN. of MEP cells Also to understand the importance of each RAR regarding metastatic dissemination and tumor growth, we generated LM38-LP cell lines stably transfected with shRNA against RARa, RARb or RARg. These cells were inoculated into the mammary fat pad of syngeneic BALB/c mice. Ten days post-inoculation, when tumors were palpable, mice received a pellet with or without ATRA (10mg/mouse) during 3 weeks and we analyzed tumor volume and number and size of spontaneous lung metastasis. ATRA treatment markedly decreased the tumor volume and the number of spontaneous metastasis in those mice inoculated with LM38-LP control cells. Tumors and/or metastases grown from cells transfected with shRNA against RARa, b or g did not respond to ATRA. Interestingly the absence of RARb led to a total loss of the metastatic capacity, suggesting the involvement of RARb in malignant progression. On the contrary, loss of RARa and RARg decreased orthotopic growth but promoted lung metastases formation, suggesting that these RARs could participate in the prevention of metastatic dissemination. In sum our in vitro studies allow us to conclude that the LEP compartment of LM38-LP cell line responds to ATRA by a classical pathway through RARb. On the other hand, ATRA modulation of the adhesiveness of LM38-LP cells to FN could be mediated by the MEP component. In vivo experiments demonstrated that the three retinoic receptors are implicated, but in a different manner, both in tumor growth and in the colonization of the target organ. 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 339.

Characterization of a Novel Primary Mammary Tumor Cell Line Reveals that Cyclin D1 Is Regulated by the Type I Insulin-Like Growth Factor Receptor

The importance of type I insulin-like growth factor receptor (IGF-IR) overexpression in mammary tumorigenesis was recently shown in two separate transgenic models. One of these models, the MTB-IGFIR transgenics, was generated in our lab to overexpress IGF-IR in mammary epithelial cells in a doxycycline (Dox)-inducible manner. To complement this transgenic model, primary cells that retained Dox-inducible expression of IGF-IR were isolated from a transgenic mammary tumor. This cell line, RM11A, expressed high levels of IGF-IR, phosphorylated Akt, and phosphorylated extracellular signal-regulated kinase 1/2 in the presence of Dox. IGF-IR overexpression provided the primary tumor cells with a survival advantage in serum-free media and seemed to induce ligand-independent activation of the IGF-IR because RM11A cells cultured in the presence of Dox were largely nonresponsive to exogenous IGFs. IGF-IR overexpression also augmented the growth of RM11A cells in vivo because injection of these cells into mammary glands of wild-type mice produced palpable tumors in 15.8 +/- 3.4 days when the mice were administered Dox, compared with 57.8 +/- 6.3 days in the absence of Dox. DNA microarray analysis revealed a number of genes regulated by IGF-IR, one of which was cyclin D1. Suppression of IGF-IR expression in vitro or in vivo was associated with a decrease in cyclin D1 protein, suggesting that at least some of the proliferative actions of IGF-IR are mediated through cyclin D1. Therefore, this article characterizes the first primary murine mammary tumor cell line with inducible IGF-IR expression. These cells provide a powerful in vitro/in vivo model to examine the function of IGF-IR in mammary tumorigenesis.

Rosiglitazone inhibits metastasis development of a murine mammary tumor cell line LMM3

BackgroundActivation of peroxisome proliferator-activated receptors γ (PPARγ) induces diverse effects on cancer cells. The thiazolidinediones (TZDs), such as troglitazone and ciglitazone, are PPARγ agonists exhibiting antitumor activities; however, the underlying mechanism remains inconclusive. Rosiglitazone (RGZ), a synthetic ligand of PPARγ used in the treatment of Type 2 diabetes, inhibits growth of some tumor cells and is involved in other processes related to cancer progression. Opposing results have also been reported with different ligands on tumor cells. The purpose of this study was to determine if RGZ and 15d-PGJ2 induce antitumor effects in vivo and in vitro on the murine mammary tumor cell line LMM3.MethodsThe effect on LMM3 cell viability and nitric oxide (NO) production of different doses of RGZ, 15-dPGJ2, BADGE and GW9662 were determined using the MTS colorimetric assay and the Griess reaction respectively. In vivo effect of orally administration of RGZ on tumor progression was evaluated either on s.c. primary tumors as well as on experimental metastasis. Cell adhesion, migration (wound assay) and invasion in Transwells were performed. Metalloproteinase activity (MMP) was determined by zymography in conditioned media from RGZ treated tumor cells. PPARγ expression was detected by inmunohistochemistry in formalin fixed tumors and by western blot in tumor cell lysates.ResultsRGZ orally administered to tumor-bearing mice decreased the number of experimental lung metastases without affecting primary s.c. tumor growth. Tumor cell adhesion and migration, as well as metalloproteinase MMP-9 activity, decreased in the presence of 1 μM RGZ (non-cytotoxic dose). RGZ induced PPARγ protein expression in LMM3 tumors. Although metabolic activity -measured by MTS assay- diminished with 1–100 μM RGZ, 1 μM-treated cells recovered their proliferating capacity while 100 μM treated cells died. The PPARγ antagonist Biphenol A diglicydyl ether (BADGE) did not affect RGZ activity. On the contrary, the specific antagonist GW9662 completely abrogated RGZ-induced decrease in cell viability. A decrease in NO levels was detected in the presence of either 1 or 100 μM RGZ. The natural ligand 15d-PGJ2 did not affect metabolic activity although it induced a significant decrease in NO production.ConclusionA significant decrease in the number of experimental LMM3 lung metastasis, but not on primary tumor growth, after oral RGZ administration was observed. In vitro, 100 μMRGZ also reduced cell viability and NO production, while no changes were observed in the presence of 15d-PGJ2. BADGE did not reverse RGZ effect while the antagonist GW9662 completely abrogated it, suggesting a PPARγ- dependent mechanism. Inhibition of lung metastatic nodules by RGZ administered in vivo, might be associated with the observed decrease in MMP-9 expression, in cell adhesion, migration and invasion. RGZ augmented its expression. PPARγ was detected in cell lysates by western blot and by immunohistochemistry in tumors from RGZ-treated mice. In summary we can suggest that RGZ or any other TZDs might be possible future approaches in the treatment of metastasis of PPARγ-expressing cells.

Identification and transcript analysis of a novel wallaby (Macropus eugenii) basal-like breast cancer cell line

BackgroundA wide variety of animal models have been used to study human breast cancer. Murine, feline and canine mammary tumor cell lines have been studied for several decades and have been shown to have numerous aspects in common with human breast cancer. It is clear that new comparative approaches to study cancer etiology are likely to be productive.ResultsA continuous line of breast carcinoma cells (WalBC) was established from a primary breast cancer that spontaneously arose in a female tammar wallaby (Macropus eugenii). The primary tumor was 1.5 cm3 and although large, did not appear to invade the stroma and lacked vimentin expression. The WalBC cell line was cultured from the primary tumor and passaged for 22 months. WalBC cells displayed an epithelial morphology when grown on plastic, were not EGF responsive, stained strongly for cyto-keratin and negatively for vimentin. WalBC cells were shown to be non-invasive within a Matrigel invasion assay and failed to produce tumors following transplantation into nude mice. Gene expression profiling of WalBC cells was performed using a cDNA microarray of nearly 10,000 mammary gland cDNA clones and compared to normal primary mammary cells and profiles of human breast cancer. Seventy-six genes were down-regulated and sixty-six genes were up-regulated in WalBC cells when compared to primary mammary cells. WalBC cells exhibited expression of known markers of basal invasive human breast cancers as well as increased KRT17, KRT 14 and KRT 19, DSP, s100A4, NDRG-1, ANXA1, TK1 and AQP3 gene expression and decreased gene expression of TIMP3, VIM and TAGLN. New targets for breast cancer treatment were identified such as ZONAB, PACSIN3, MRP8 and SUMO1 which have human homologues.ConclusionThis study demonstrates how novel models of breast cancer can provide new fundamental clues regarding cancer etiology which may lead to new human treatments and therapies.

Osteopontin Regulates Ubiquitin-Dependent Degradation of Stat1 in Murine Mammary Epithelial Tumor Cells

Background: Osteopontin (OPN) is a secreted glycoprotein that mediates cell-matrix interactions and cellular signaling by binding with integrin (primarily αvβ3) and CD44 receptors. OPN regulates cell adhesion, chemotaxis, macrophage-directed IL-10 suppression, stressdependent angiogenesis, apoptosis prevention, anchorage-independent growth of tumor cells. However, the molecular mechanisms that define the role of OPN in tumor progression and metastasis are incompletely understood. Methods: In this study, we use a system of 4T1 and 4T07 murine mammary epithelial tumor cell lines that are divergent in both metastatic phenotype and OPN expression. 4T1 expresses OPN and hematogeneously metastasizes, whereas 4T07 does not express OPN and is highly tumorigenic but fails to metastasize. Results: Our results demonstrate that OPN regulates Stati protein degradation through the ubiquitin-proteasome pathway to alter interferon-γ-dependent growth inhibition and p21 expression. We identify Stat-interacting LIM protein as the critical Stat ubiquitin E3 ligase in this setting. Conclusions: OPN regulates Stati-dependent functions, such as growth inhibition and p21 expression, in the murine mammary epithelial cells lines 4T1 and 4T07. This relationship between OPN and Stati in the context of tumor biology has not been previously examined.