Mouse Mammary Tumor Cells Research Articles

Abstract 2555: ZC3H8 associates with PML bodies and influences aggressive tumor cell behavior

Abstract The ZC3H8 gene encodes a protein with three zinc finger motifs in the C-terminal region, suggesting a role as an RNA binding protein, but whose specific function remains unclear. ZC3H8 protein localizes to both PML bodies and Cajal bodies within the nucleus, as confocal microscopy demonstrates colocalization of ZC3H8 with both PML and coilin proteins. ZC3H8 has a casein kinase 2 phosphorylation site in the N-terminal region, and treatment with a casein kinase 2 inhibitor causes the numerous PML bodies and their associated ZC3H8 to coalesce to a few larger bodies. Removal of the inhibitor restores PML bodies to their original state. We constructed a mutant of ZC3H8 that removed the predicted CK2 phosphorylation site by replacing the serine with an alanine residue. Cells transfected with this mutant had reduced numbers of PML bodies, similar to cells treated with the CK2 inhibitor. In contrast, a mutant constructed with a glutamic acid in place of the phosphorylatable serine was predicted to behave as a constitutive mutant. This mutant did not exhibit differences from wild type localization. These experiments suggest that ZC3H8 integrity is key to maintenance of PML bodies. Other studies have identified ZC3H8 as a component of the LEC (little elongation complex), and we found that another identified component, ICE2 (Narg) also localizes to PML bodies. The ZC3H8 gene is overexpressed in a number of human and mouse breast cancer cell lines, and elevated mRNA levels are associated with a poorer prognosis for women with breast cancer. We have used RNA silencing to decrease levels of expression in two independent mouse mammary tumor cell lines. Cells with lower ZC3H8 expression have dramatic phenotypic changes, showing decreased rates of proliferation, slower migration in wound healing assays, formation of fewer and smaller colonies in soft agar assays, and decreased ability to invade through a basement membrane in transwell assays. Further, ZC3H8-depleted cells have decreased rates of growth as spheroids and decreased growth in vivo in syngeneic mice. We developed an inducible vector driving expression of ZC3H8. Upon treatment with the inducer, doxycycline, cells assume a more aggressive phenotype. We introduced a synonymous mutant of ZC3H8 unable to be silenced by the RNAi into the knockdown cells, and rescued the aggressive phenotype of the original tumor cells. Overexpression of ZC3H8 in non-tumorigenic COMMA-D cells leads to a less pronounced, but opposite, increase in proliferation, motility, and invasion. We have engineered the synonymous ZC3H8 with disrupted zinc finger motifs, and preliminary results suggest that disruption of the first zinc finger is not sufficient to cause loss of the aggressive phenotype. We suggest that ZC3H8 is an oncogene with substantial effects on the cancer cell phenotype. These effects are hypothesized to be associated with ZC3H8 function in PML bodies. Citation Format: John A. Schmidt, Emily Duffner, Gerard Walker, Keith G. Danielson, Janice E. Knepper. ZC3H8 associates with PML bodies and influences aggressive tumor cell behavior [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2555. doi:10.1158/1538-7445.AM2017-2555

MicroRNAs-143 and -145 induce epithelial to mesenchymal transition and modulate the expression of junction proteins.

Transforming growth factor (TGF)-β is one of the major inducers of epithelial to mesenchymal transition (EMT), a crucial program that has a critical role in promoting carcinoma's metastasis formation. MicroRNAs-143 and -145, which are both TGF-β direct transcriptional targets, are essential for the differentiation of vascular smooth muscle cells (VSMC) during embryogenesis, a TGF-β-dependent process reminiscent of EMT. Their role in adult tissues is however less well defined and even ambiguous, as their expression was correlated both positively and negatively with tumor progression. Here we show that high expression of both miRs-143 and -145 in mouse mammary tumor cells expressing constitutively active STAT3 (S3C) is involved in mediating their disrupted cell-cell junctions. Additionally, miR-143 appears to have a unique role in tumorigenesis by enhancing cell migration in vitro and extravasation in vivo while impairing anchorage-independent growth, which may explain the contradictory reports about its role in tumors. Accordingly, we demonstrate that overexpression of either miRNA in the non-transformed mammary epithelial NMuMG cells leads to upregulation of EMT markers and of several endogenous TGF-β targets, downmodulation of a number of junction proteins and increased motility, correlating with enhanced basal and TGF-β-induced SMAD-mediated transcription. Moreover, pervasive transcriptome perturbation consistent with the described phenotype was observed. In particular, the expression of several transcription factors involved in the mitogenic responses, of MAPK family members and, importantly, of several tight junction proteins and the SMAD co-repressor TGIF was significantly reduced. Our results provide important mechanistic insight into the non-redundant role of miRs-143 and -145 in EMT-related processes in both transformed and non-transformed cells, and suggest that their expression must be finely coordinated to warrant optimal migration/invasion while not interfering with cell growth.

OVOL2 antagonizes TGF-β signaling to regulate epithelial to mesenchymal transition during mammary tumor metastasis.

Great progress has been achieved in the study of the role of TGF-β signaling in triggering epithelial-mesenchymal transition (EMT) in a variety of cancers; however, the regulation of TGF-β signaling during EMT in mammary tumor metastasis has not been completely defined. In the present study, we demonstrated that OVOL2, a zinc finger transcription factor, inhibits TGF-β signaling-induced EMT in mouse and human mammary tumor cells, as well as in mouse tumor models. Data from the Oncomine databases indicated a strong negative relationship between OVOL2 expression and breast cancer progression. Moreover, our experiments revealed that OVOL2 inhibits TGF-β signaling at multiple levels, including inhibiting Smad4 mRNA expression and inducing Smad7 mRNA expression, blocking the binding between Smad4 and target DNA, and interfering with complex formation between Smad4 and Smad2/3. These findings reveal a novel mechanism that controls the TGF-β signaling output level in vitro and in vivo. The modulation of these molecular processes may represent a strategy for inhibiting breast cancer invasion by restoring OVOL2 expression.

The transcription factor MAFK induces EMT and malignant progression of triple-negative breast cancer cells through its target GPNMB.

Triple-negative breast cancer (TNBC) is particularly aggressive and difficult to treat. For example, the transforming growth factor-β (TGF-β) pathway is implicated in TNBC progression and metastasis, but its opposing role in tumor suppression in healthy tissues and early-stage lesions makes it a challenging target. Therefore, additional molecular characterization of TNBC may lead to improved patient prognosis by informing the development and optimum use of targeted therapies. We found that musculoaponeurotic fibrosarcoma (MAF) oncogene family protein K (MAFK), a member of the small MAF family of transcription factors that are induced by the TGF-β pathway, was abundant in human TNBC and aggressive mouse mammary tumor cell lines. MAFK promoted tumorigenic growth and metastasis by 4T1 cells when implanted subcutaneously in mice. Overexpression of MAFK in mouse breast epithelial NMuMG cells induced epithelial-mesenchymal transition (EMT) phenotypes and promoted tumor formation and invasion in mice. MAFK induced the expression of the gene encoding the transmembrane glycoprotein nmb (GPNMB). Similar to MAFK, GPNMB overexpression in NMuMG cells induced EMT, tumor formation, and invasion, in mice, whereas knockdown of MAFK in tumor cells before implantation suppressed tumor growth and progression. MAFK and GPNMB expression correlated with poor prognosis in TNBC patients. These findings suggest that MAFK and its target gene GPNMB play important roles in the malignant progression of TNBC cells, offering potentially new therapeutic targets for TNBC patients.

Artesunate Enhances the Cytotoxicity of 5-Aminolevulinic Acid-Based Sonodynamic Therapy against Mouse Mammary Tumor Cells In Vitro.

Sonodynamic therapy (SDT) kills tumor cells through the synergistic effects of ultrasound (US) and a sonosensitizer agent. 5-Aminolevulinic acid (5-ALA) has been used as a sonodynamic sensitizer for cancer treatment. However, studies have shown that 5-ALA-based SDT has limited efficacy against malignant tumors. In this study, we examined whether artesunate (ART) could enhance the cytotoxicity of 5-ALA-based SDT against mouse mammary tumor (EMT-6) cells in vitro. In the ART, ART + US, ART + 5-ALA, and ART + 5-ALA + US groups, the cell survival rate correlated with ART concentration, and decreased with increasing concentrations of ART. Morphologically, many apoptotic and necrotic cells were observed in the ART + 5-ALA + US group. The percentage of reactive oxygen species-positive cells in the ART + 5-ALA + US group was also significantly higher than that in the 5-ALA group (p = 0.0228), and the cell death induced by ART + 5-ALA + US could be inhibited by the antioxidant N-acetylcysteine. These results show that ART offers great potential in enhancing the efficacy of 5-ALA-based SDT for the treatment of cancer. However, these results are only based on in vitro studies, and further in vivo studies are required.

Buformin inhibits the stemness of erbB-2-overexpressing breast cancer cells and premalignant mammary tissues of MMTV-erbB-2 transgenic mice

BackgroundMetformin, an FDA-approved drug for the treatment of Type II diabetes, has emerged as a promising anti-cancer agent. Other biguanide analogs, including buformin and phenformin, are suggested to have similar properties. Although buformin was shown to reduce mammary tumor burden in carcinogen models, the anti-cancer effects of buformin on different breast cancer subtypes and the underlying mechanisms remain unclear. Therefore, we aimed to investigate the effects of buformin on erbB-2-overexpressing breast cancer with in vitro and in vivo models.MethodsMTT, cell cycle, clonogenic/CFC, ALDEFLUOR, tumorsphere, and Western blot analyses were used to determine the effects of buformin on cell growth, stem cell populations, stem cell-like properties, and signaling pathways in SKBR3 and BT474 erbB-2-overexpressing breast cancer cell lines. A syngeneic tumor cell transplantation model inoculating MMTV-erbB-2 mice with 78617 mouse mammary tumor cells was used to study the effects of buformin (1.2 g buformin/kg chow) on tumor growth in vivo. MMTV-erbB-2 mice were also fed buformin for 10 weeks, followed by analysis of premalignant mammary tissues for changes in morphological development, mammary epithelial cell (MEC) populations, and signaling pathways.ResultsBuformin significantly inhibited SKBR3 and BT474 cell growth, and in vivo activity was demonstrated by considerable growth inhibition of syngeneic tumors derived from MMTV-erbB-2 mice. In particular, buformin suppressed stem cell populations and self-renewal in vitro, which was associated with inhibited receptor tyrosine kinase (RTK) and mTOR signaling. Consistent with in vitro data, buformin suppressed mammary morphogenesis and reduced cell proliferation in MMTV-erbB-2 mice. Importantly, buformin decreased MEC populations enriched with mammary reconstitution units (MRUs) and tumor-initiating cells (TICs) from MMTV-erbB-2 mice, as supported by impaired clonogenic and mammosphere formation in primary MECs. We further demonstrated that buformin-mediated in vivo inhibition of MEC stemness is associated with suppressed activation of mTOR, RTK, ER, and β-catenin signaling pathways.ConclusionsOverall, our results provide evidence for buformin as an effective anti-cancer drug that selectively targets TICs, and present a novel prevention and/or treatment strategy for patients who are genetically predisposed to erbB-2-overexpressing breast cancer.

MMTV-PyMT and Derived Met-1 Mouse Mammary Tumor Cells as Models for Studying the Role of the Androgen Receptor in Triple-Negative Breast Cancer Progression.

Triple-negative breast cancer (TNBC) has a faster rate of metastasis compared to other breast cancer subtypes, and no effective targeted therapies are currently FDA-approved. Recent data indicate that the androgen receptor (AR) promotes tumor survival and may serve as a potential therapeutic target in TNBC. Studies of AR in disease progression and the systemic effects of anti-androgens have been hindered by the lack of an AR-positive (AR+) immunocompetent preclinical model. In this study, we identified the transgenic MMTV-PyMT (mouse mammary tumor virus-polyoma middle tumor-antigen) mouse mammary gland carcinoma model of breast cancer and Met-1 cells derived from this model as tools to study the role of AR in breast cancer progression. AR protein expression was examined in late-stage primary tumors and lung metastases from MMTV-PyMT mice as well as in Met-1 cells by immunohistochemistry (IHC). Sensitivity of Met-1 cells to the AR agonist dihydrotestosterone (DHT) and anti-androgen therapy was examined using cell viability, migration/invasion, and anchorage-independent growth assays. Late-stage primary tumors and lung metastases from MMTV-PyMT mice and Met-1 cells expressed abundant nuclear AR protein, while negative for estrogen and progesterone receptors. Met-1 sensitivity to DHT and AR antagonists demonstrated a reliance on AR for survival, and AR antagonists inhibited invasion and anchorage-independent growth. These data suggest that the MMTV-PyMT model and Met-1 cells may serve as valuable tools for mechanistic studies of the role of AR in disease progression and how anti-androgens affect the tumor microenvironment.

Fetoprotein Derived Short Peptide Coated Nanostructured Amphiphilic Surfaces for Targeting Mouse Breast Cancer Cells

In this work, self-assembled tumor targeting nanostructured surfaces were developed from a newly designed amphiphile by conjugating boc protected isoleucine with 2,[Formula: see text] ethylenedioxy bis ethylamine (IED). To target mouse mammary tumor cells, a short peptide sequence derived from the human alpha-fetoprotein (AFP), LSEDKLLACGEG was attached to the self-assembled nanostructures. Tumor targeting and cell proliferation were examined in the presence of nanoscale assemblies. To further obliterate mouse breast tumor cells, the chemotherapeutic drug tamoxifen was then entrapped into the nanoassemblies. Our studies indicated that the targeting systems were able to efficiently encapsulate and release tamoxifen. Cell proliferation studies showed that IED-AFP peptide loaded with tamoxifen decreased the proliferation of breast cancer cells while in the presence of the IED-AFP peptide nanoassemblies alone, the growth was relatively slower. In the presence of human dermal fibroblasts however cell proliferation continued similar to controls. Furthermore, the nanoscale assemblies were found to induce apoptosis in mouse breast cancer cells. To examine live binding interactions, SPR analysis revealed that tamoxifen encapsulated IED-AFP peptide nanoassemblies bound to the breast cancer cells more efficiently compared to unencapsulated assemblies. Thus, we have developed nanoscale assemblies that can specifically bind to and target tumor cells, with increased toxicity in the presence of a chemotherapeutic drug.

Spontaneous mouse mammary tumor cell lysates induce IgG production in spleen mononuclear cells of healthy and tumor-bearing mice

ABSTRACTBackground. We hypothesized that Tumor cell lysate (TCL) prepared from spontaneous mouse mammary tumor (SMMT) may elicit IgG production by spleen mononuclear cells (SMCs) in ex vivo.Methods. The SMCs from healthy mice (HM, n = 6) and four-week SMMT-bearing mice (TBM, n = 6) was cultured in presence of TCL and mitogen for 42 hr at 37°C, separately. Serum and SMCs culture supernatant levels of IFNγ, IL-4, and total IgG were measured using special ELISA kits.Results. Serum IgG level of TBM was significantly higher than that of HM group (P = 0.019), while serum IFNγ and IL-4 did not differ between two groups (P > 0.05). Mitogen significantly induced ex vivo production of both IFNγ (P = 0.013) and IL-4 (P = 0.015) by SMCs from HM group, and only IL-4 (P = 0.049) by SMCs from TBM group. In contrast, TCL increased ex vivo production of IgG by SMCs from both HM (P = 0.034) and TBM (P = 0.016) groups. The ex vivo IgG revealed a moderate positive correlation with tumor size (r = 0.578, P = 0.422).Conclusion. It seems that TCL prepared from SMMT are potent inducers of IgG production. This may propose TCL as a potential tool for monitoring of humoral immunity in animal models.

Abstract 4321: The pro-tumorigenic effects of obesity are reversed by severe weight loss via chronic or intermittent calorie restriction but not weight normalization via a low-fat diet

Abstract Background: Breast cancer outcomes in both pre- and postmenopausal women are negatively impacted by obesity, but the reversibility of these pro-cancer effects via weight loss has not been well established. We examined how weight loss via four different dietary interventions affects mammary tumor progression in a mouse model of chronic obesity and pre-menopausal basal-like breast cancer. Methods: Six week old C57BL/6 mice were randomized to a control (Con, 10% kcal from fat) or diet-induced obesity (DIO, 60% kcal from fat) regimen and maintained on these diets for 15 weeks. DIO mice were then randomized to remain on DIO diet (Obese) or begin one of four weight loss regimens: low-fat control (formerly obese, FOb), high-carb 30% calorie-restricted (HCCR), low-carb 30% CR (LCCR), or intermittent energy restriction (IER). IER mice received a 14% CR diet 5 days/week and a 70% CR diet on 2 non-consecutive days/week, such that their average daily kcal intake and macronutrient distribution was equivalent to the LCCR group. The Con mice received the same control diet through the end of the study to maintain a normal weight. Mice were orthotopically injected with E0771 mouse mammary tumor cells at week 25. All mice were euthanized when one tumor reached 1.5 cm in diameter. Results: At study endpoint, Obese mice had a higher average body weight and body fat percentage in comparison to all other diet groups. These phenotypic measures were statistically equivalent in Con and FOb mice, indicating that the FOb mice had returned to a normal weight and body fat percentage, but they were significantly lower in the 3 CR groups. The CR mice all lost and then maintained an equivalent amount of weight following the diet switch at 15 weeks, regardless of the diet timing or macronutrient distribution. Tumor volume and weight at sacrifice were significantly greater in the Obese mice relative to Con mice and all CR mice, but not FOb mice, despite their weight loss to Con levels. In contrast, tumors in the HCCR and IER, but not LCCR, mice were significantly smaller than Con. Serum insulin and leptin levels were statistically equivalent in Con, FOb, HCCR, LCCR, and IER, but serum insulin-like growth factor 1 remained significantly elevated in FOb mice, with levels statistically equivalent to Obese mice. Conclusions: These results suggest that weight normalization via a low-fat control diet does not reverse the mammary tumor-promoting effects of chronic obesity. However, severe weight loss through various forms of calorie restriction successfully reverses these effects. An improved understanding of the energy responsive mechanisms mediating the anticancer effects of calorie restriction in obese mice may lead to the identification of new intervention targets and strategies to reduce the impact of obesity on breast cancer outcomes. Citation Format: Laura W. Bowers, Emily L. Rossi, Meghana G. Shamsunder, Stephen D. Hursting. The pro-tumorigenic effects of obesity are reversed by severe weight loss via chronic or intermittent calorie restriction but not weight normalization via a low-fat diet. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4321.

Abstract 748: Targeting the crosstalk between tumor cells and adipocytes to block breast cancer progression

Abstract It is now well-established that the local microenvironment of an emerging tumor plays a vital role in various steps of tumorigenesis. Stromal cells, such as fibroblasts, endothelial and various inflammatory cells, can contribute to tumor progression, and tumor cells can directly model their microenvironment, influencing host cells to secrete factors that favor cancer cell survival and growth. Recent studies have demonstrated the existence of cross-talk between breast cancer (BC) cells and adipocytes, the most abundant stromal cell in the breast, supporting their involvement in BC and consistent with epidemiological studies reporting the association of obesity and insulin resistance/diabetes with a higher risk and poor prognosis of BC. Based on this evidence, we investigated whether adipocyte de-differentiation is the crucial trigger in inducing pro-tumor microenvironment remodeling and tumor progression. Co-culture of 4T1 mouse mammary tumor cells using Transwell or their conditioned medium with mature adipocytes derived from the 3T3-L1 mouse cells induced adipocyte de-differentiation, observed as a reduced expression of specific differentiation markers (peripilin, adipocyte protein 2 (aP2), PPARã and adiponectin) in Western blots and by qRT-PCR, and a reduced triglyceride content as assessed by oil red O staining compared to mature adipocytes. Strong adipocyte de-differentiation, associated with an increase in pro-inflammatory molecules (IL6 and CCL2), was also induced in vitro by tumor interstitial fluids obtained from MMT-PyMT transgenic mice, but not by plasma of these mice. Preliminary studies to identify molecules involved in the cross-talk indicated that the phenomenon is exosome-unrelated. Based on the relevant role of the PPARã transcription factor in inducing and maintaining adipocyte differentiation, we co-cultured adipocytes with 4T1 BC cells in the presence of several PPARã agonists enriched in the diets of many populations displaying a low incidence of cancer (þ3-fatty acids), and in the presence of glitazones (rosiglitazone and pioglitazone) to force adipocyte stable differentiation. While most of these compounds did not block adipocyte de-differentiation induced by tumor cells, pioglitazone and eicosanoid acid showed some activity in inhibiting this de-differentiation in vitro. Treatment of MMTV-PyMT transgenic mice at tumor onset by oral gavage with pioglitazone did not significantly reduce mammary tumor growth compared to controls, whereas eicosanoid acid treatment significantly reduced tumor multiplicity and tumor burden compared to controls. Overall, our results suggest that tumor-induced adipocyte de-differentiation participates in tumorigenesis and that the blockade of this program can inhibit tumor progression. Supported by AIRC and Fondazione Cariplo. Citation Format: Tiziana Triulzi, Valentina Ciravolo, Viola Regondi, Martina Di Modica, Claudia Chiodoni, Elda Tagliabue. Targeting the crosstalk between tumor cells and adipocytes to block breast cancer progression. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 748.

Serotonin transporter antagonists target tumor-initiating cells in a transgenic mouse model of breast cancer.

Accumulating data suggests that the initiation and progression of human breast tumors is fueled by a rare subpopulation of tumor cells, termed breast tumor-initiating cells (BTIC), which resist radiotherapy and chemotherapy. Consequently, therapies that abrogate BTIC activity are needed to achieve durable cures for breast cancer patients. To identify such therapies we used a sensitive assay to complete a high-throughput screen of small molecules, including approved drugs, with BTIC-rich mouse mammary tumor cell populations. We found that inhibitors of the serotonin reuptake transporter (SERT) and serotonin receptors, which include approved drugs used to treat mood disorders, were potent inhibitors of mouse BTIC activity as determined by functional sphere-forming assays and the initiation of tumor formation by transplant of drug-exposed tumor cells into syngeneic mice. Moreover, sertraline (Zoloft), a selective serotonin reuptake inhibitor (SSRI), synergized with docetaxel (Taxotere) to shrink mouse breast tumors in vivo. Hence drugs targeting the serotonergic system might be repurposed to treat breast cancer patients to afford more durable breast cancer remissions.

Abstract 2017: The CEBPD transcription factor, a marker of good prognosis in breast cancer, becomes a signaling hub for promotion of cancer cell stemness by hypoxia and interleukin-6

Abstract The transcription factor C/EBPdelta (CEBPD) is expressed in normal mammary epithelial cells and high expression in breast cancer correlates with hormone receptors and good prognosis (Mendoza-Villanueva et al., submitted). In the MMTV-Neu mouse model, deletion of CEBPD increases tumor multiplicity, but “paradoxically” reduces metastatic progression, suggesting that CEBPD has dual tumor suppressing and metastasis promoting functions (Balamurugan et al., 2010). Here, we identify a molecular mechanism for the tumor promoting function of CEBPD that may reach beyond breast cancer. We show that CEBPD serves as a signaling hub that integrates and amplifies pathways that promote stemness in breast cancer, glioblastoma and embryonic cancer cells. This work identifies a mechanism by which hypoxia and inflammation promote cancer stem-like cells (CSCs), which are implicated in tumor progression and resistance. Our earlier studies showed that CEBPD promotes inflammatory signaling and hypoxia adaptation by inhibiting the expression of FBXW7 (Balamurugan et al., 2010 and 2013), which can target several oncoproteins for degradation. Using a variety of established breast and glioblastoma tumor cell lines/xenografts, primary mouse mammary tumor cells and tissues, recent patient-derived glioblastoma cell lines, as well as embryonic stem/cancer cell lines, we find that deletion or depletion of CEBPD reduced the number of stem cells as determined by surface markers, sphere formation, limiting dilution xenografts, and by expression of stemness genes. Mechanistic studies in culture models show that CEBPD is expressed in CSCs and connects and amplifies both hypoxia (HIF-1) and inflammation (IL-6/STAT3) pathways to increase Notch1/NICD expression and cell stemness. CEBPD induces NICD expression transcriptionally through HIF-1 and at the level of the protein by inhibition of FBXW7-mediated degradation. In addition to its known role as a target of STAT3 and activator of IL-6 gene expression, we identified an additional role for CEBPD in promoting expression of the IL-6 receptor. Furthermore, we found that CEBPD also directly activates the expression of stemness-promoting genes such as OCT4, Sox2, KLF4, Myc, Nanog, CD44 and CD133. Taken together, this study provides novel insights into the molecular mechanisms that promote cancer cell stemness. Because CEBPD integrates and amplifies multiple stemness promoting pathways, CEBPD may represent a unique point of vulnerability in cancer stem cells, strongly suggesting that pharmacological inhibition of CEBPD signaling may effectively target CSCs. Results from current efforts to pharmacologically downregulate CEBPD in cancer stem cells will also be presented. Citation Format: Kuppusamy Balamurugan, Daniel Mendoza-Villanueva, Barbara Rath, Philip Tofilon, Shikha Sharan, Esta Sterneck. The CEBPD transcription factor, a marker of good prognosis in breast cancer, becomes a signaling hub for promotion of cancer cell stemness by hypoxia and interleukin-6. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2017.

CCL5 activation of CCR5 regulates cell metabolism to enhance proliferation of breast cancer cells.

In earlier studies, we showed that CCL5 enhances proliferation and survival of MCF-7 breast cancer cells in an mTOR-dependent manner and we provided evidence that, for T cells, CCL5 activation of CCR5 results in increased glycolysis and enhanced ATP production. Increases in metabolic activity of cancer cells, specifically increased glycolytic activity and increased expression of glucose transporters, are associated with tumour progression. In this report, we provide evidence that CCL5 enhances the proliferation of human breast cancer cell lines (MDA-MB-231, MCF-7) and mouse mammary tumour cells (MMTV-PyMT), mediated by CCR5 activation. Concomitant with enhanced proliferation we show that CCL5 increases cell surface expression of the glucose transporter GLUT1, and increases glucose uptake and ATP production by these cells. Blocking CCL5-inducible glucose uptake abrogates the enhanced proliferation induced by CCL5. We provide evidence that increased glucose uptake is associated with enhanced glycolysis, as measured by extracellular acidification. Moreover, CCL5 enhances the invasive capacity of these breast cancer cells. Using metabolomics, we demonstrate that the metabolic signature of CCL5-treated primary mouse mammary tumour cells reflects increased anabolic metabolism. The implications are that CCL5–CCR5 interactions in the tumour microenvironment regulate metabolic events, specifically glycolysis, to promote tumour proliferation and invasion.

Obesity-Associated Alterations in Inflammation, Epigenetics, and Mammary Tumor Growth Persist in Formerly Obese Mice.

Using a murine model of basal-like breast cancer, we tested the hypothesis that chronic obesity, an established breast cancer risk and progression factor in women, induces mammary gland epigenetic reprogramming and increases mammary tumor growth. Moreover, we assessed whether the obesity-induced epigenetic and protumor effects are reversed by weight normalization. Ovariectomized female C57BL/6 mice were fed a control diet or diet-induced obesity (DIO) regimen for 17 weeks, resulting in a normal weight or obese phenotype, respectively. Mice on the DIO regimen were then randomized to continue the DIO diet or were switched to the control diet, resulting in formerly obese (FOb) mice with weights comparable with control mice. At week 24, all mice were orthotopically injected with MMTV-Wnt-1 mouse mammary tumor cells. Mean tumor volume, serum IL6 levels, expression of proinflammatory genes in the mammary fat pad, and mammary DNA methylation profiles were similar in DIO and FOb mice and higher than in controls. Many of the genes found to have obesity-associated hypermethylation in mice were also found to be hypermethylated in the normal breast tissue of obese versus nonobese human subjects, and nearly all of these concordant genes remained hypermethylated after significant weight loss in the FOb mice. Our findings suggest that weight normalization may not be sufficient to reverse the effects of chronic obesity on epigenetic reprogramming and inflammatory signals in the microenvironment that are associated with breast cancer progression. Cancer Prev Res; 9(5); 339-48. ©2016 AACR.

Abstract B29: Dissecting the effects of Brd4 short isoform on breast cancer progression

Abstract Breast cancer is one of the most common female malignancies in the United States with metastatic disease being the main contributor to breast cancer morbidity and mortality. Our lab previously identified BRD4, a member of the bromodomain family, as a regulator of breast cancer progression. Brd4 is a chromatin-modifying factor that regulates the expression of a large set of genes. Brd4 is normally expressed as two isoforms. The long isoform (Brd4-LF) contains two bromodomains in the N-terminus, an ET domain, a serine-rich SEED domain, and a proline-rich C-terminus. The short isoform (Brd4-SF) differs from the long isoform by a deletion of the proline-rich C-terminus and a three amino acid difference on the N-terminal domain. Previous studies in our laboratory indicate that these two isoforms have opposing effects on breast cancer progression. Highly metastatic mouse mammary tumor cells overexpressing Brd4-LF suppressed tumor growth and dissemination, whereas overexpression of Brd4-SF increased metastatic outgrowth in lungs. We have shown that these two isoforms are found in different compartments within the cell nucleus, leading to altered interaction with nuclear proteins. Brd4-LF is localized in the nuclear matrix while Brd4-SF is found in the nuclear envelope where it interacted with the LINC complex and known metastasis susceptibility proteins RRP1B and SIPA1. Given that these two isoforms reside in different subnuclear locations, we hypothesize that the isoforms bind to different sets of genes. To test this hypothesis we are utilizing the chromatin-immunoprecipitation sequencing (ChIP-seq) assay to characterize the genetic interactions associated with Brd4-SF. Identification of the Brd4-SF interacting genes will provide us with mechanistic insight into the underlying causes of the different metastatic outcomes promoted by the two isoforms, highlighting potential targets for future targeted therapies. Citation Format: Xinyi Zhu, Kent W. Hunter. Dissecting the effects of Brd4 short isoform on breast cancer progression. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr B29.

Abstract B12: Polymorphisms in the Arntl2 promoter affect metastatic susceptibility in breast cancer

Abstract Metastasis accounts for the majority of cancer-related deaths. Although exact mechanisms are unknown, studies in our laboratory have demonstrated that germline polymorphisms could alter tumorigenesis and metastasis. Our lab developed a mouse genetics approach to investigate the effect of inherited single nucleotide polymorphisms (SNPs) on metastasis by crossing inbred mice of various backgrounds and subsequent mapping of quantitative trait loci (QTL) responsible for the phenotype. Using this approach, we crossed a highly metastatic (FVB) and low metastatic (MOLF) mouse strain and identified Arntl2, a circadian rhythm transcription factor, as a gene with differential expression that predicted distant metastasis-free survival in breast cancer patients. shRNA-mediated reduction in Arntl2 expression suppressed lung metastases, establishing Arntl2 as a metastasis modifier. Initial analysis revealed no polymorphisms in coding regions that could account for the differential expression in FVB and MOLF. Therefore, we performed whole genome sequencing of MOLF to identify SNP differences between MOLF and FVB. Overlapping this analysis with DNAse hypersensitivity sites in mouse mammary tumor cell lines revealed 13 polymorphisms in the predicted promoter region of Arntl2. FVB and MOLF promoters exhibited differential promoter activity in vitro, suggesting that these SNPs could explain the difference in Arntl2 expression between these two strains. In order to validate the causative role of the SNPs in vivo, metastatic cell lines were engineered using CRISPR technology to specifically replace the FVB with the MOLF promoter. Substitution of this promoter reduced Arntl2 transcript levels and subsequently decreased pulmonary metastases, as expected. Since Arntl2 is a transcription factor, current studies are focused on identifying Arntl2-regulated genes to investigate the pathways that are involved in modulating metastasis. This study will have important implications regarding the function of circadian rhythm in cancer progression and provide a potential molecular mechanism to explain the increased risk of breast cancers in nightshift workers. Furthermore, to the best of our knowledge this would be the first example of genome editing to validate polymorphisms in transcriptional control elements associated with inherited susceptibility for metastasis. Citation Format: Ngoc-Han Ha, Kent W. Hunter. Polymorphisms in the Arntl2 promoter affect metastatic susceptibility in breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr B12.

Diverse roles of cell-specific hypoxia-inducible factor 1 in cancer-associated hypercoagulation

AbstractDespite the increased risk of thrombosis in cancer patients compared with healthy individuals, mechanisms that regulate cancer-induced hypercoagulation are incompletely understood. The aim of this study was to investigate whether cell-specific hypoxia-inducible factor (HIF) 1α regulates cancer-associated hypercoagulation, using in vitro clotting assays and in vivo cancer models. In mouse lung and mammary tumor cells, hypoxia led to increases in cell adhesion, clotting, and fibrin deposition; these increases were eliminated in HIF1α null cells. Increased levels of HIF1α were also associated with increased tissue factor expression in human breast tumor samples. Conversely, deletion of endothelial (but not myeloid) cell-specific HIF1α doubled pulmonary fibrin deposition, and trebled thrombus formation compared with wildtype littermates in tumor-bearing mice. Our data suggest that tumor and endothelial cell-specific HIF1α may have opposing roles in cancer-associated coagulation and thrombosis. Off-target effects of manipulating the HIF1 axis in cancer patients should be carefully considered when managing thrombotic complications.

Tuning cell adhesion on polymeric and nanocomposite surfaces: Role of topography versus superhydrophobicity

Development of surface modification procedures which allow tuning the cell adhesion on the surface of biomaterials and devices is of great importance. In this study, the effects of different topographies and wettabilities on cell adhesion behavior of polymeric surfaces are investigated. To this end, an improved phase separation method was proposed to impart various wettabilities (hydrophobic and superhydrophobic) on polypropylene surfaces. Surface morphologies and compositions were characterized by scanning electron microscopy and X-ray photoelectron spectroscopy, respectively. Cell culture was conducted to evaluate the adhesion of 4T1 mouse mammary tumor cells. It was found that processing conditions such as drying temperature is highly influential in cell adhesion behavior due to the formation of an utterly different surface topography. It was concluded that surface topography plays a more significant role in cell adhesion behavior rather than superhydrophobicity since the nano-scale topography highly inhibited the cell adhesion as compared to the micro-scale topography. Such cell repellent behavior could be very useful in many biomedical devices such as those in drug delivery and blood contacting applications as well as biosensors.

Abstract P2-05-28: The impact of the plant lignin secoisolariciresinol diglycoside on preclinical models of estrogen receptor positive breast cancer

Abstract Background: Several preclinical studies indicate that secoisolariciresinol diglycoside (SDG), a polyphenolic plant lignin found most abundantly in flaxseeds, inhibits the progression of both estrogen receptor (ER) positive and negative mammary tumors. SDG is metabolized by gut bacteria into the biologically active metabolites enterolactone (ENL) and enterodiol (END), which are known to have anti-estrogenic activity. However, the mechanisms mediating SDG's anti-tumor effects remain poorly understood. Methods: In a dose-determination pilot study linked to an ongoing clinical trial of SDG in women at high risk for breast cancer, 18 week old C57BL/6 mice were randomized to a control diet or SDG-supplemented diets (25 or 74 mg/kg of food) for 8 weeks prior to euthanization, and the levels of serum and tissue SDG metabolites (particularly ENL and END), metabolic hormones and inflammatory markers were measured. In an ongoing tumor study, 12-week old C57BL/6 and foxn1 nu/nu mice were randomized to the control or control plus SDG (100 mg/kg of food, a dose projected to match ENL and END metabolite levels achieved in the clinical trial) diet regimen. After 8 weeks on diet, they will receive orthotopic injections of E0771 mouse mammary tumor cells or BT-483 human breast cancer cells (both ER positive), continuing on the same diets until euthanization. Cell culture studies examining the impact of biologically relevant concentrations of ENL and END on E0771 and BT-483 cells are also in progress. Results: In comparison to those maintained on the control diet, the higher dose SDG diet reduced estrogen and pro-inflammatory signaling in the pilot study mice, as evidenced by higher interleukin 10 and lower C-reactive protein mammary fat pad expression as well as lower circulating levels of the adipokines leptin and resistin, which have been linked to chronic inflammation. High dose SDG also decreased serum insulin and glucose levels, indicating improved metabolic function. Because serum ENL and END levels in the pilot study did not reach those achieved in the SDG clinical trial, a 100 mg/kg SDG dose was chosen for the tumor study. Cell culture studies indicate that ENL (150 nM) inhibits E0771 and BT-483 cell proliferation and ER alpha:beta expression ratio. Conclusions: Preliminary data suggests that the anti-tumor effects of SDG's metabolites may be mediated through multiple mechanisms, including improvements in metabolic function and inflammatory signaling as well as modulation of breast cancer cell gene expression. The results of the ongoing tumor study will inform the design of additional cell culture studies aimed at further defining these mechanisms. Citation Format: Bowers LW, Ford NA, Rossi EL, Shamsunder MG, Kimler BF, Fabian CJ, Hursting SD. The impact of the plant lignin secoisolariciresinol diglycoside on preclinical models of estrogen receptor positive breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P2-05-28.