Mouse Mammary Tumor Cell Line Research Articles

Abstract 251: MCAM controls mammary cell state in luminal progenitor-derived breast cancer and alveologenesis

Abstract To establish and maintain functional and properly formed tissues, cells must integrate cues from their environment into fate decisions. External cues—from other cells and the extracellular matrix for instance—must be met with appropriate responses to ensure cell fate decisions that control lineage plasticity and favor proper development, maintenance, or regeneration of tissues while suppressing neoplastic transformation. MCAM (CD146; MUC18; S-endo-1; and Gicerin) is an integral membrane glycoprotein whose misexpression is associated with a variety of epithelial cancers, including breast cancer. However, the mechanisms by which MCAM conveys extracellular cues into malignant cellular outputs is not well understood. Our observation that MCAM is normally expressed in transplantable fetal and adult mammary stem cells (MaSCs) with multilineage potential, suggested it could control mammary cell state plasticity in both neoplastic and normal mammary tissue. Using a plastic, mouse mammary tumor cell line derived from the MMTV-PyMT mouse (Py230) that has been reported to exhibit mammary stem cell and luminal progenitor features, we tested the role of Mcam in controlling the tumorigenicity and lineage/cell-state relationships of mammary tumor cells. We found that Mcam knockdown in Py230 cells increases STAT3 expression, activation, and downstream target transcription, leading to a shift in the predominant cell state away from a luminal progenitor state toward more differentiated alveolar and basal phenotypes. Mechanistically, these changes involve altered activity of casein kinase II (CK2) against multiple substrates including STAT3 and PTEN. This effect may be mediated by direct coupling and sequestration of CK2 to various substrates via the cytoplasmic tail of MCAM. We have also found that inhibition of Ck2 or Stat3 reversed the transcriptional state change elicited by Mcam knockdown. In grafted tumors in vivo, this state switch correlates with reduced overall tumorigenicity and attenuated expression of Sox10, a gatekeeper of neural crest-like invasive features. In the normal gland, we hypothesize that this activity regulates multilineage stem cell potential upon transplantation and multilineage regenerative potential across lactation/involution cycles as we found reciprocal expression patterns for MCAM and STAT3 in alveolar mammary epithelial cells. These data point to an integral role for MCAM in lineage plasticity of normal mammary epithelial cells that is co-opted during transformation to potentiate tumor cell plasticity, progression, and evasion of targeted cancer therapies. Citation Format: Brooke L. Gates, Ozlen Balcioglu, David W. Freeman, Berhane M. Hagos, Benjamin T. Spike. MCAM controls mammary cell state in luminal progenitor-derived breast cancer and alveologenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 251.

Abstract 5364: Progesterone receptor modulates the antigen processing and presentation machinery decreasing MHC class I expression on tumors

Abstract Hormone receptor (HR) positive breast cancers, which account for approximately 75% of all breast cancers and express both estrogen receptor (ER) and progesterone receptor (PR), have been the subject of much research. As such, successful anti-estrogen/ER-based therapies have been very successful in the treatment of HR+ breast cancer. Despite this success, over 1/3 of patients will eventually progress on current ER/estrogen-targeted therapies, underscoring the need for new targeted therapies in HR+ breast cancer. Unlike ER, the role of PR in breast cancer progression has received far less attention. In a previous study, we found that a mouse mammary tumor cell line E0771, modified to express the mouse progesterone receptor (mPR) and the OVA peptide, demonstrated decreased T cell-mediated cell death when treated with progesterone compared to the control group. Progesterone-mediated protection from T-cell death was only observed in mPR+ cell lines. Flow analysis of the E0771-Ova-mPR cells also revealed a decrease in MHC Class I presentation in progesterone-treated cells compared to a vehicle control. To further explore the role of the machinery required for antigen processing and presentation of MHC Class I molecules, we conducted additional experiments using the E0771 cell line. Our results showed that treatment with progesterone led to a reduction in RNA levels of all the machinery required for antigen processing and presentation (APP). Based on these findings, we hypothesize that activation of the progesterone receptor by progesterone may decrease MHC Class I expression by regulating APP. Our results showed that treatment with progesterone resulted in a reduction in RNA levels of Tap1, Tap2, Tapbp, Nlrc5, B2m, and Psmb8, which are essential for APP. We therefore propose that activation of the progesterone receptor by progesterone may decrease MHC Class I expression by directly modulating the APP pathway. The findings of this study may have significant implications for our understanding of how breast cancer cells evade T-cell mediated cytotoxicity. Citation Format: Julio Tinoco, Lauryn Werner, Eilidh Chowanec, Harmony Saunders, Sun Xiaopeng, Jay Gertz, Justin Balko, Zachary C. Hartman, Christy Hagan. Progesterone receptor modulates the antigen processing and presentation machinery decreasing MHC class I expression on tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5364.

Antiproliferative effect of methanolic extract of Tamarindus indica in C127I cell line

Investigation of natural bioactive compounds that possess antioxidant and anticancer properties have drawn interest in scientific and industrial perspectives. The current study was conducted to assess the antiproliferative action of methanolic extract of seed coat of Tamarindus indica in C127I mouse mammary tumour cell line. 3-(4,5-dimethyl thazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay is performed to assess the cytotoxicity of methanolic extracts of T. indica in the C127I cell line at concentrations of 320, 160, 80, 40, 20, and 10 µg/mL. The half maximal inhibitory concentration (IC50) was then calculated using Graph Pad Prism 5.0. Doxorubicin was used as positive control. After determining IC50, the cells were treated with extract of T. indica at half, IC50 and double IC50 concentrations after being seeded at a concentration of 1x105 cells/mL in 6 well plates. After 24 hours the cells were trypsinised and stained with acridine orange-ethidium bromide (AOEB) to assess the morphology of apoptosis. When the cells were exposed to various doses of the extract, a dose-dependent decrease in cell viability was observed, and the IC50 value of T. indica was estimated to be 13 µg/ml. In the control cells, AO/EB labelling revealed proliferating cells with green fluorescence, but the T. indica extract-treated cells displayed dose dependent shift from orange to red fluorescence. In conclusion, methanolic extract of Tamarindus indica was found to possess antiproliferative effect in C127I cell line comparable to doxorubicin.

Functional and Phenotypic Characterisations of Common Syngeneic Tumour Cell Lines as Estrogen Receptor-Positive Breast Cancer Models

Estrogen receptor-positive breast cancers (ER+ BCas) are the most common form of BCa and are increasing in incidence, largely due to changes in reproductive practices in recent decades. Tamoxifen is prescribed as a component of standard-of-care endocrine therapy for the treatment and prevention of ER+ BCa. However, it is poorly tolerated, leading to low uptake of the drug in the preventative setting. Alternative therapies and preventatives for ER+ BCa are needed but development is hampered due to a paucity of syngeneic ER+ preclinical mouse models that allow pre-clinical experimentation in immunocompetent mice. Two ER-positive models, J110 and SSM3, have been reported in addition to other tumour models occasionally shown to express ER (for example 4T1.2, 67NR, EO771, D2.0R and D2A1). Here, we have assessed ER expression and protein levels in seven mouse mammary tumour cell lines and their corresponding tumours, in addition to their cellular composition, tamoxifen sensitivity and molecular phenotype. By immunohistochemical assessment, SSM3 and, to a lesser extent, 67NR cells are ER+. Using flow cytometry and transcript expression we show that SSM3 cells are luminal in nature, whilst D2.0R and J110 cells are stromal/basal. The remainder are also stromal/basal in nature; displaying a stromal or basal Epcam/CD49f FACS phenotype and stromal and basal gene expression signatures are overrepresented in their transcript profile. Consistent with a luminal identity for SSM3 cells, they also show sensitivity to tamoxifen in vitro and in vivo. In conclusion, the data indicate that the SSM3 syngeneic cell line is the only definitively ER+ mouse mammary tumour cell line widely available for pre-clinical research.

Abstract 950: Role of epithelial-mesenchymal plasticity in immune remodeling during metastasis

Abstract Introduction: Breast cancer is the most prevalent type of cancer in women, accounting for 12% of all annual cancer cases worldwide with most deaths attributed to metastasis. It displays both inter- and intra-tumoral heterogeneity which is linked to epithelial-mesenchymal transition (EMT). This program involves the acquisition of a mesenchymal-like phenotype allowing cells to disseminate from the primary tumor and metastasize. However, recent studies show that tumor cells do not need to undergo complete EMT to gain invasive potential; instead, co-expression of both epithelial and mesenchymal markers is critical for metastasis. Such tumor cells are thought to display epithelial-mesenchymal plasticity (EMP) which bestows them with enhanced fitness and flexibility to fulfill diverse requirements of the metastatic cascade. Additionally, hybrid EM phenotype has been linked to immunesuppression but has been understudied due to lack to suitable models with intact immune system. Thus, this necessitates the mechanistic dissection of tumor immune microenvironment (TIME) in models with EM plasticity. To characterize the contribution of EMP to metastasis, we utilized syngeneic mouse mammary tumor cell lines, which are hybrid EM and investigated their proteomic and cytokine profile and identified several novel cytokines. Methods: Reverse Phase Protein Array (RPPA) was performed to determine the proteomic profile of tumors with EM plasticity. In addition, Luminex assay was performed to assess the cytokine/chemokine profile in above tumor models. Results: The RPPA involved a panel of 450 antibodies and showed that tumors with EM plasticity were enriched in specific immunesuppressive proteins. At the same time, Luminex analysis was suggestive of recruitment of tumor-associated macrophages (TAMs) in models with EM plasticity. Additionally, cells with EM plasticity showed the ability to induce tumor-promoting/M2-like macrophages (in vitro). Conclusions: Our data indicates that cells/tumors with EM plasticity show differential protein expression than E and M counterparts and are enriched in specific immunesuppressive proteins. Moreover, such cells have immune networks (cytokines/chemokines) that promote recruitment of TAMs - the largest myeloid infiltrate in the microenvironment of solid tumors. Thus, our study invites more attention to investigate the mechanisms of immunesuppression and metastasis in tumors with EM plasticity. Citation Format: Tanvi H. Visal. Role of epithelial-mesenchymal plasticity in immune remodeling during metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 950.

Abstract 1531: CD146 couples and sequesters regulatory kinases to control breast cancer cell state plasticity

Abstract CD146 (Mcam; Muc18; s-endo; and gicerin) is an integral membrane glycoprotein originally identified in a human malignant melanoma, but mis-expressed in a wider variety of malignancies including breast cancer, where it has been implicated in controlling tumor growth and dissemination. The mechanisms by which CD146 conveys extracellular cues into malignant cellular outputs is not well understood, but its high-level expression in early development and normal mouse mammary stem cells, as well as expression in highly plastic adult pericytes (formerly known as mesenchymal stem cells) suggested to us that it may control cell state plasticity. We tested the role of CD146 in a plastic, stem cell-like mouse mammary tumor cell line derived from the MMTV-PyMT mouse (PY230). We identified domains in the CD146 cytoplasmic tail essential for controlling substrate utilization by upstream kinases in Akt, Mapk, Stat3, and integrin-related signaling pathways that alter cell state plasticity and tumorigenicity. This work suggests that MCAM plays a key role in controlling lineage plasticity in normal and neoplastic mammary cells with relevance for understanding tumor cell plasticity, progression and evasion of targeted cancer therapies. Citation Format: Ozlen Balcioglu, Brooke L. Gates, David W. Freeman, Berhane M. Hagos, Benjamin T. Spike. CD146 couples and sequesters regulatory kinases to control breast cancer cell state plasticity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1531.

Synthesis and molecular modeling of MetAP2 of thiosemicarbazides, 1,2,4-triazoles, thioethers derived from (S)-Naproxen as possible breast cancer agents

New thiosemicarbazides (3, 5-6), 1,2,4-triazoles (14-15) and thioethers (22-68) from derived (S)-Naproxen were synthesized in this study. The structure of these compounds were elucidated by spectral (FT-IR, 1H NMR, 13C NMR) methods, besides elemental analysis and TLC. The molecular binding of the compounds on MetAP-2 was performed. Anticancer effects of the synthesized compounds were studied by using MTT assay method on MCF-7 (includes oestrogene and progesterone receptors) and MDA-MB-231 (lacks estrogen and progesterone receptors) adenocarcinoma cell lines at 0, 10, 25, 50, 75 and 100 μM concentrations for 24 h. The IC50 values of novel (S)-Naproxen derivatives were determined between from 5 to 100 μM on MCF-7 breast cancer cell line and MDA-MB-231 cell lines. The apoptotic activity of selected compounds 22 and 42 were first analyzed by Annexin V staining using Tali Image-Based Cytometer. Mitochondrial membrane potential changes determined in fluorescence plate reader following JC-1 stain for compounds 22 and 42 in MCF-7 and MDA-MB-231 cells. The effect of these compounds on the cell viability 4T1 mouse mammary tumor cell line was tested at 1 to 5 times of calculated IC50 value (IC50x1, IC50x2, IC50x3, IC50x4, and IC50x5). Next in order to determine the toxicity of the combination of compound 51 and Docetaxel, WST-1 cell viability and proliferation assay was performed with 4T1.

STAT5 is activated in macrophages by breast cancer cell-derived factors and regulates macrophage function in the tumor microenvironment

BackgroundIn breast cancer, complex interactions between tumor cells and cells within the surrounding stroma, such as macrophages, are critical for tumor growth, progression, and therapeutic response. Recent studies have highlighted the complex nature and heterogeneous populations of macrophages associated with both tumor-promoting and tumor-inhibiting phenotypes. Defining the pathways that drive macrophage function is important for understanding their complex phenotypes within the tumor microenvironment. Signal transducer and activator of transcription (STAT) transcription factors, such as STAT5, are key regulators of immune cell function. The studies described here investigate the functional contributions of STAT5 to tumor-associated macrophage function in breast cancer.MethodsInitial studies were performed using a panel of human breast cancer and mouse mammary tumor cell lines to determine the ability of tumor cell-derived factors to induce STAT5 activation in macrophages. Further studies used these models to identify soluble factors that activate STAT5 in macrophages. To delineate STAT5-specific contributions to macrophage function, a conditional model of myeloid STAT5 deletion was used for in vitro, RNA-sequencing, and in vivo studies. The effects of STAT5 deletion in macrophages on tumor cell migration and metastasis were evaluated using in vitro co-culture migration assays and an in vivo tumor cell-macrophage co-injection model.ResultsWe demonstrate here that STAT5 is robustly activated in macrophages by tumor cell-derived factors and that GM-CSF is a key cytokine stimulating this pathway. The analysis of RNA-seq studies reveals that STAT5 promotes expression of immune stimulatory genes in macrophages and that loss of STAT5 in macrophages results in increased expression of tissue remodeling factors. Finally, we demonstrate that loss of STAT5 in macrophages promotes tumor cell migration in vitro and mammary tumor metastasis in vivo.ConclusionsBreast cancer cells produce soluble factors, such as GM-CSF, that activate the STAT5 pathway in macrophages and drive expression of inflammatory factors. STAT5 deletion in myeloid cells enhances metastasis, suggesting that STAT5 activation in tumor-associated macrophages protects against tumor progression. Understanding mechanisms that drive macrophage function in the tumor microenvironment will ultimately lead to new approaches that suppress tumor-promoting functions while enhancing their anti-tumor functions.

​Identification of Suitable Reference Genes for qPCR Analysis of 4T1 Mouse Mammary Tumor Cell Line

Background: Identification of candidate reference genes for real time PCR study is a preliminary requirement to normalize experimental data and thus, deduce a reliable conclusion. Complex tissues like mouse mammary gland constitutes various cell types which makes it difficult to identify reference gene constantly expressing under different experimental conditions. Methods: In this study we have identified suitable reference genes for 4T1 tumor cell line derived from mouse mammary tumor cells. We have studied four genes namely Gapdh, Actb, Prdx1 and Ctbp1 for their expression stability in CPV2.NS1 post transfected 4T1 cells by Best Keeper. Result: By our study, three reference genes i.e. Prdx1, Gapdh and Ctbp1 were found to be quite correlated with the BestKeeper index, but by considering all three criteria of selection by BestKeeper algorithm, Prdx1 showed minimum standard deviation and coefficient of variation and was found to be ranked at first position by BestKeeper which suggests Prdx1 to be considered as better internal control gene among all other reference genes taken in our study for qPCR based experiments in 4T1 mouse mammary tumor cell line transfected with CPV2.NS1

Chk1 suppression leads to a reduction in the enhanced radiation-induced invasive capability on breast cancer cells.

Radiation therapy is generally effective for treating breast cancers. However, approximately 30% of patients with breast cancer experience occasional post-treatment local and distant metastasis. Low-dose (0.5 Gy) irradiation is a risk factor that promotes the invasiveness of breast cancers. Although an inhibitor of checkpoint kinase 1 (Chk1) suppresses the growth and motility of breast cancer cell lines, no study has investigated the effects of the combined use of a Chk1 inhibitor and radiation on cancer metastasis. Here, we addressed this question by treating the human breast cancer cell line MDA-MB-231 (in vitro) and mouse mammary tumor cell line 4 T1 (in vitro and in vivo) with γ-irradiation and the Chk1 inhibitor PD407824. Low-dose γ-irradiation promoted invasiveness, which was suppressed by PD407824. Comprehensive gene expression analysis revealed that low-dose γ-irradiation upregulated the mRNA and protein levels of S100A4, the both of which were downregulated by PD407824. We conclude that PD407824 suppresses the expression of S100A4. As the result, γ-irradiation-induced cell invasiveness were inhibited.

Combination Therapy of Breast Cancer by Codelivery of Doxorubicin and Survivin siRNA Using Polyethylenimine Modified Silk Fibroin Nanoparticles.

Here, polyethylenimine (PEI) modified silk fibroin nanoparticles (SFNPs) were prepared for codelivery of doxorubicin (DOX) and survivin siRNA. The prepared NPs were characterized in terms of stability and structural, functional, and physicochemical properties. Moreover, the ability of the conjugate to escape from the endosome and cellular uptake were assessed. Afterward, the in vivo therapeutic efficacy was analyzed in the mice model. The siRNA loaded PEI-SFNPs showed acceptable size, zeta potential, and stability in serum. It also effectively induced apoptosis in the 4T1 mouse mammary tumor cell line. Cellular uptake and endosomal escape analyses confirmed that PEI-SFNPs containing siRNA could escape from the endosome and accumulate in the cytoplasm of 4T1 cells. Real time-PCR indicated the significant decrease in the expression of survivin mRNA in the 4T1 cell line 48 h postincubation with siRNA loaded PEI-SFNPs. In vivo biodistribution of PEI-SFNPs confirmed higher accumulation of SFNPs in the tumor site compared with other organs. The codelivery systems remarkably reduced the growth rate of breast tumor in the mice model without any obvious weight lost. Histopathological and tunnel staining exhibited more apoptotic tumor cells in the group containing both DOX and survivin siRNA. Tumorigenic breast tissue resected from the animals after treatment with siRNA also exhibited significant suppression of survivin gene. In conclusion, the prepared drug delivery system had an acceptable potential in tumor removal, apoptosis induction in cancer cells, and therapeutic efficacy. Thus, it would be a good candidate for breast cancer therapy.

Intracellular microRNA expression patterns influence cell death fates for both necrosis and apoptosis.

MicroRNAs (miRNAs) are small noncoding RNA molecules that interact with target mRNAs at specific sites to induce cleavage of the mRNA or inhibit translation. Such miRNAs play a vital role in gene expression and in several other biological processes, including cell death. We have studied the mechanisms regulating cell death (necrosis in original F28-7 cells and apoptosis in their variant F28-7-A cells) in the mouse mammary tumor cell line FM3A using the anticancer agent floxuridine (FUdR). We previously reported that inhibition of heat-shock protein 90 by the specific inhibitor geldanamycin (GA) in F28-7 cells causes a shift from necrosis to apoptosis. In this study, we investigated the intracellular miRNA expression profiles of FUdR-treated F28-7 cells (necrotic condition), GA plus FUdR-treated F28-7 cells (apoptotic condition), and FUdR-treated F28-7-A cells (apoptotic condition) through miRNA microarray analysis. In addition, we knocked down Dicer, a key molecule for the expression of mature miRNAs, in F28-7 cells to examine whether it modulates FUdR-induced cell death. Our analysis revealed that the miRNA expression patterns differ significantly between these cell death conditions. Furthermore, we identified miRNA candidates that regulate cell death. Knockdown of Dicer in FUdR-treated necrosis-fated cells caused a partial shift from necrosis to apoptosis. These findings suggest that modulation of miRNA expression patterns influences the decision of cell death fate toward necrosis or apoptosis. Our findings may serve as a basis for further study of the functions of miRNAs in cell death mechanisms.

Effect of Biotin-Targeted Protein-Based Nanoparticles Contain of Curcumin on the Expression of Apoptotic Index Bax and Bcl2 Proteins

In this study, biotin functionalized bovine serum albumin (BSA-biotin) loaded with curcumin (CUR), was synthesized and evaluated as drug carrier. The aim of this study is evaluation of the curcumin induced apoptosis related proteins, Bcl-2 and Bax expression via targeted albumin based nanoparticles. The synthesized BSA-biotin-CUR was characterized by different techniques such as: TEM, UV–Vis, FTIR, and DLS. The loading efficiency of CUR was 7.9%. The result indicates that the particles were measured around 185.4 nm with a zeta potential of -16.8 mV. Cytotoxicity results on mouse mammary tumor cell line (4T1) confirmed that formulated CUR, BSA-biotin-CUR, has high anticancer effect compared to free CUR. Also, real-time PCR method revealed that BSA-biotin-CUR increased Bax protein expression, and also downregulated Bcl-2 protein in 4T1 cells, which proven the ability of synthesized nanoparticles. We suggest that BSA-biotin-CUR can improve therapeutic effect, of CUR so that it can overcome the limitations of CUR with a great potential for further evaluations and applications.

Obesity-Associated Leptin Signaling Promotes Chemotherapy Resistance in Basal-Like Breast Cancer: The Role of Tumor-Associated Macrophages

Abstract Objectives Obesity is associated with a reduced response to cytotoxic chemotherapies. Given that the adipokine leptin has been shown to promote macrophage production of pro-tumor cytokines, we hypothesized that leptin-induced pro-tumor macrophage polarization is a key mediator of obesity-associated docetaxel resistance. Methods Wild-type C57BL/6 mice were fed a low-fat control (10% kcal from fat) or high-fat diet-induced obesity (DIO; 60% kcal from fat) diet for 15 weeks, then orthotopically injected with 2 mouse mammary tumor cell lines: EWnt-S (scrambled shRNA) and EWnt-L (shRNA to the leptin receptor) in the 9th and 4th mammary glands, respectively. Mice in each group were then randomized to vehicle or docetaxel (20 mg/kg/week IV for 3 weeks). The transcriptomes of both tumors (n = 6/group) were analyzed by microarray, and cell type enrichment analysis performed using the webtool xCell. The in vitro effects of leptin +/− docetaxel on macrophage polarization as well as cancer cell cytotoxicity and markers of invasive capacity are currently being assessed. Results The DIO mice had greater body weight and % body fat at euthanization versus control mice (P < 0.01 for both). Docetaxel treatment in DIO mice reduced tumor growth rate in EWnt-L tumors (P < 0.05), but not EWnt-S tumors. In contrast, docetaxel treatment reduced the EWnt-L and EWnt-S tumor growth rate in control mice (P < 0.05 for both). xCell analysis indicated that docetaxel treatment increased M2 macrophage levels in the DIO mouse tumors, but not the controls. Leptin receptor knockdown attenuated this effect. In addition, key genes related to “macrophage markers”, “cytokines and inflammatory response”, and “interleukin (IL)-6 signaling” were differentially expressed in docetaxel-treated EWnt-S tumors from DIO versus control mice. Conclusions These results indicate that leptin signaling mediates obesity-induced docetaxel resistance in the E-Wnt model of basal-like breast cancer. They also suggest that docetaxel interacts with leptin signaling to promote an increase in tumor-promoting M2 macrophages and subsequent docetaxel resistance. This hypothesis is currently being explored through our in vitro studies. Funding Sources This work was supported by a grant from the Developmental Funding Program, Lineberger Comprehensive Cancer Center, UNC Chapel Hill.

SAT-137 The Effect of Hypertriglyceridemia on Triple Negative Breast Cancer Progression

Obesity is associated with increased cancer risk and cancer-associated mortality1,2. Hypertriglyceridemia (HTG), a component of the metabolic syndrome which frequently co-exists with obesity, has been associated with increased breast cancer risk and mortality in triple negative breast cancer (TNBC)3,4. To determine if HTG is causally related to enhanced TNBC progression in the absence of other obesity-associated characteristics, TNBC growth and metastasis in a mouse model of HTG was examined. Mice overexpressing human apolipoprotein C3 (AC3) were backcrossed onto FVB/N background and crossed with recombination-activating gene 1 (Rag1) knockout mice to generate immunodeficient HTG mice. AC3 mice relative to wild-type (WT) littermates showed a 20-fold higher circulating triglycerides (p < 0.0001) and elevated very low density lipoprotein (VLDL) cholesterol (p = 0.001). No differences in body weight, body composition, blood glucose or plasma insulin levels were observed between the two groups, allowing for investigation on the influence of HTG on TNBC without confounders such as hyperinsulinemia or hyperglycemia. AC3 mice orthotopically implanted with the mouse mammary tumor cell line, Mvt1, showed both increased tumor growth (AC3 vs WT: 1157.0 ± 84.2 vs 707.2 ± 58.6 mm3, p = 0.0009) and lung metastasis (AC3 vs WT: 57.3 ± 3.0 vs 32.9 ± 5.3 mm3, p = 0.001) relative to WT mice. Immunodeficient Rag1/AC3 mice likewise, showed increased tumor growth compared to WT controls when implanted with human TNBC MDA-MB-231 cells (AC3 vs WT: 363.2 ± 113.9 vs 92.95 ± 16.2 mm3, p = 0.038). To investigate how HTG affects tumor lipid metabolism, serum and tumors from both groups were analyzed by liquid chromatography/mass spectrometry. Total alkyl-acyl, di-acyl-phosphatidylcholines and sphingomyelin concentrations were higher in the serum of AC3 mice relative to WT. In contrast, no overall difference in tumor phospholipid or acylcarnitine content was noted between AC3 and WT mice, suggesting no difference in fatty acid oxidation in the setting of HTG. Mvt1 tumors from AC3 and WT mice were analyzed by RNA sequencing. Decreased expression of genes associated with cholesterol synthesis (Fdft1, Pvmk, Acss2) were found in tumors from AC3 mice. Tumors from AC3 mice also showed decreased protein expression of LDLR, which is associated with LDL cholesterol uptake. Overall, these findings suggest that HTG, independently of other obesity-associated characteristics such as hyperinsulinemia and hyperglycemia, leads to changes in intracellular lipid metabolism and promotes TNBC progression.

Establishment and Characterization of a Brca1-/-, p53-/- Mouse Mammary Tumor Cell Line.

Breast cancer is the most commonly occurring cancer in women and the second most common cancer overall. By the age of 80, the estimated risk for breast cancer for women with germline BRCA1 or BRCA2 mutations is around 80%. Genetically engineered BRCA1-deficient mouse models offer a unique opportunity to study the pathogenesis and therapy of triple negative breast cancer. Here we present a newly established Brca1−/−, p53−/− mouse mammary tumor cell line, designated as CST. CST shows prominent features of BRCA1-mutated triple-negative breast cancers including increased motility, high proliferation rate, genome instability and sensitivity to platinum chemotherapy and PARP inhibitors (olaparib, veliparib, rucaparib and talazoparib). Genomic instability of CST cells was confirmed by whole genome sequencing, which also revealed the presence of COSMIC (Catalogue of Somatic Mutations in Cancer) mutation signatures 3 and 8 associated with homologous recombination (HR) deficiency. In vitro sensitivity of CST cells was tested against 11 chemotherapy agents. Tumors derived from orthotopically injected CST-mCherry cells in FVB-GFP mice showed sensitivity to cisplatin, providing a new model to study the cooperation of BRCA1-KO, mCherry-positive tumor cells and the GFP-expressing stromal compartment in therapy resistance and metastasis formation. In summary, we have established CST cells as a new model recapitulating major characteristics of BRCA1-negative breast cancers.

Abstract 3681: RAD51AP1 is a novel therapeutic target for cancer prevention and treatment

Abstract Although much progress has been made in recent years in its treatment and prevention, cancer is still the second leading cause of death in the United States. Surgical removal of the tumor is not possible in all cancer types; therefore, chemotherapy and radiation therapy have become the standard course of treatment and are often the only option for more late stage and metastatic tumors. Unfortunately, chemotherapy and radiation therapy resistance are the greatest challenge for physicians trying to eradicate disease, prevent tumor recurrence, and inhibit distant metastasis. This resistance is derived from a heterogeneous population of cells within the tumor known as cancer stem cells (CSCs). CSCs are able to maintain a higher capacity for self-renewal due to a more efficient DNA repair system. CSCs are actively cycling, therefore, the repair mechanism of homologous recombination (HR) plays a significant role in repairing double-stranded DNA breaks that inevitably accumulate. Several studies have examined the link between efficient DNA repair and CSC self-renewal and found that proteins involved in HR repair are elevated in many human cancers. RAD51-associated protein 1 (RAD51AP1), which is responsible for the successful resolution of HR during DNA repair, is overexpressed in wide variety of human cancers. The present study sought to determine the functional role of RAD51AP1 in CSC self-renewal and its relevance to cancer growth and progression and also drug resistance. Our studies provide evidence that RAD51AP1 plays a critical role in CSC self-renewal and maintenance in breast, lung, and colon cancers. To determine the functional role of RAD51AP1 in cancer growth and progression, we generated genetically engineered mouse (GEM) models in breast and lung in WT (Rad51ap1+/+) and Rad51ap1-knockout (KO, Rad51ap1-/-) background and found that Rad51ap1 deletion significantly delayed the time of tumor formation and distant metastasis, which in turn increase the overall survival. Rad51ap1 inactivation in human breast and lung cancer cell lines, significantly reduced tumor growth in xenograft mouse model. This findings are also recapitulated in mouse mammary tumor cell lines (AT3 and 4T1). We also investigated the functional role of RAD51AP1 on colon cancer growth and progression using AOM/DSS and Apcmin/+ models of colon cancer and found that smaller tumor burden in KO mice compared to WT suggesting that Rad51ap1 may have a significant role in tumor growth. Taken together, these data demonstrate that RAD51AP1 plays a critical role in CSC growth and self-renewal in many human cancers and RAD51AP1 could be a novel therapeutic target for cancer prevention and treatment. Citation Format: Allison E. Bridges, Pragya Rajpurohit, Parth Patel, Nagendra Singh, Putter D. Prasad, Muthusamy Thangaraju. RAD51AP1 is a novel therapeutic target for cancer prevention and treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3681.

Abstract 3845: Anti-tumor potential of Hydralazine, a DNMT1 inhibitor, on breast cancer growth and progression

Abstract Breast cancer (BC) is the second largest diagnosed cancer among women, with approximately 266,120 new cases of BC in the United States. Thus far BC is understood as a disease driven by genetic abnormalities. Specifically, recent studies provide evidence that epigenetic modifications, such as methylation profiles, play a critical role in BC development regarding tumor suppressor genes. Despite there being many DNMT inhibitors, such as 5- azacytidine (Vidaza), they are ineffective versus solid tumors and they elicit a cytotoxic response. Thus, it is vital that a drug that is able to halt BC growth and progression is investigated. This project hopes to test the anti-tumor potential and therapeutic potential of Hydralazine (HDZ) because of its ability to act as a DNA methylation inhibitor, specifically DNMT1 and DNMT3A. In addition, despite HDZ being currently tested in clinical cancer trials for its antihypertensive effect, its mechanism of antitumor action remains undefined.We analyzed the anti-cancer potential of HDZ on two human BC cell lines and a mouse mammary tumor cell line (AT3). Cells were treated with different doses of HDZ (0, 1, 10, 25, and 50 M) for different time points (1, 2, 3, 7, and 14 days) and cell viability was measured by MTT assay. From these experiments, we optimized the time and concentration of HDZ in BC cells. Finally, we treated the cells with the appropriate concentration and time and then prepared RNA and protein for gene expression analysis, measured DNMT1 activity, and analyzed stem cells status using cell surface markers (CD24/CD44). We also tested the anti-tumor potential of HDZ in a mouse model using AT3 cells. AT3 cells were implanted in the mammary fat pads and grown to the tumor size of 250 mm3. Then, we treated the mice with HDZ and monitored the tumor growth. Tumor tissues harvested from these mice will be used for gene expression analysis, measure DNMT1 activity, histological analysis and analyze the stem cell status such as Oct4, Nanog, Gata4, Slug, and Sox2.All 4 BC cell lines demonstrated a slightly lower cell viability from the MTT when treated for 3 days and used at the highest dosage. In addition, through the use of RT-PCR, it has been shown that the levels of DNMT1 and CD24 expressions have been slightly downregulated. Western Blot analysis showed, the protein levels of DNMT1 and CD24 also decreased slightly. We also found that HDZ treatment significantly reduced tumor growth and volume in the mouse model. These findings suggest that the drug HDZ has a slight anti-tumor property on breast cancer growth and progression in the cell and mouse models. Although not as significant as we originally thought, the future implications of this project would be to complement HDZ with another drug such as a stem cell inhibitor. We believe that this dual therapy would synergistically work to halt BC progression and growth much more efficiently than any monotherapy for the treatment of triple-negative breast cancer. Citation Format: Parth B. Patel. Anti-tumor potential of Hydralazine, a DNMT1 inhibitor, on breast cancer growth and progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3845.

Abstract SY28-04: Obesity-associated leptin signaling promotes chemotherapy resistance in triple-negative breast cancer: The role of tumor-initiating cell enrichment

Abstract SY28-04: Obesity-associated leptin signaling promotes chemotherapy resistance in triple-negative breast cancer: The role of tumor-initiating cell enrichment

Abstract 4357: The lactate receptor Gpr81 on non-cancer cells promotes an immunosuppressive phenotype in the tumor microenvironment

Abstract Cancer cells display a unique phenomenon in which, even in the presence of oxygen, cells switch from oxidative phosphorylation to glycolysis as the primary source of ATP with consequent production of lactic acid. This phenomenon, called the Warburg Effect, is a hallmark of cancer. Lactic acid has long been considered as the necessary end product of this metabolic switch, where lactic acid is effluxed out of tumor cells to prevent intracellular acidification. Recent evidence however suggests that lactate and the excess protons in the tumor microenvironment play an active role in tumor growth. In particular, lactate has been shown to function as an agonist for GPR81, a G-protein-coupled receptor expressed on the surface of tumor cells. This autocrine signaling of lactate promotes tumor growth and metastasis, as well as angiogenesis and immune evasion. The present study assesses whether tumor cell-derived lactate has any paracrine role via its receptor in non-cancer cells present in the tumor microenvironment. We generated MMTV-PyMT-Tg mice, a spontaneous model for breast cancer, on Gpr81+/+ and Gpr81-/- backgrounds. The absence of Gpr81 reduced the mammary tumor incidence, delayed mammary tumor progression, and reduced lung metastasis. These data demonstrate the essential role of GPR81 in breast cancer growth and metastasis, but does not differentiate between Gpr81 in tumor cells versus Gpr81 in the tumor microenvironment. We then used the syngeneic transplant of the mouse mammary tumor cell line AT-3 into the mammary fat pads of wild type and Gpr81-/- mice to assess the involvement of Gpr81 in the microenvironment. AT-3 cells are negative for Gpr81, and therefore our model limits Gpr81 expression to non-tumor cells in the host mouse. We found the growth of transplanted tumor cells was significantly reduced in Gpr81-/- mice than in wild type mice. Preliminary RNA-sequencing transcriptome analysis of AT-3 tumors suggest an immunosuppressive function of Gpr81, where tumors grown in a Gpr81-/- background have much stronger gene expression profiles in T-cell signaling pathways. Specifically, AT-3 tumors grown in Gpr81-/- host express significantly higher levels of genes that are specific for T cells and antigen-presenting cells such as MHC-II complex molecules, T-cell co-stimulatory molecules, and transmembrane CD8. It is well established that tumor-cell derived lactate promotes tumor growth in an autocrine manner via Gpr81 expressed on tumor cells; our studies extend the tumor-promoting role of lactate beyond this autocrine function to include paracrine signaling via Gpr81 expressed on immune cells and possibly other cell types in the tumor microenvironment. Citation Format: Timothy Brown, Sabarish Ramachandran, Stefan Offermanns, Vadivel Ganapathy. The lactate receptor Gpr81 on non-cancer cells promotes an immunosuppressive phenotype in the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4357.