MCF10A Human Breast Epithelial Cells Research Articles

Anticancer activity of synthesized 5-benzyl juglone on selected human cancer cell lines.

Cancer is a malignant disease that causes millions of deaths each year worldwide. As one of the cancer therapeutic strategies, chemotherapy is a means to destroy rapidly dividing cells. The main problem with cancer chemotherapy is the lack of selectivity of conventional chemotherapeutic drugs, leading to toxicity towards normal cells. Therefore, the discovery of anticancer agents with selectivity for fast-growing cancer cells was desirable. In this study, we report the synthesis and identification of the novel 5-benzyl juglone as a potential anticancer agent with selectivity toward certain cancer cell lines. An efficient synthetic method for 5-benzyl juglone has been established. The proliferation of cancer cell lines and a normal cell line treated by the target compound were studied using an MTT assay. In addition, the cell cycle arrest and apoptosis were determined by flow cytometry. Based on the Diels-Alder (D-A) reaction between 3,6-dimethoxy benzyne intermediate with furan, further acid-catalyzed intramolecular rearrangement and CAN-mediated oxidation, a convenient synthesis of 5-benzyl juglone has been achieved with high overall yield. The results from in vitro biological evaluation indicated that the juglone derivative exhibited potent antiproliferative activity against HCT-15 human colorectal cancer cells with an IC50 value of 12.27 μM. It exerted high inhibitory activity toward MCF-7 human breast cancer cells and, to a much lesser extent, to corresponding MCF-10A human breast epithelial normal cells with the IC50 ratio (IC50 in MCF-7 divided by IC50 in MCF-10A) of 0.62. The mechanistic investigations indicated that 5-benzyl juglone could induce cell cycle arrest at the G0/G1 phase and promote apoptosis of HCT-15 cells. The apoptotic effects possibly also contributed to its higher selectivity toward cancer cells than normal cell lines.

Abstract 2327: Pyruvate carboxylase rescues MCF10A-ras cell viability in extracellular matrix detached conditions

Abstract Breast cancer is the most diagnosed cancer in women, and metastasis accounts for 90% of cancer related deaths. In order to metastasize, cancer cells must survive in conditions where they are detached from the extracellular matrix (ECM). Untransformed cells decrease energy metabolism when detached from ECM, but oncogenic transformation results in rescued energy metabolism of detached cells. Expression of the anaplerotic enzyme pyruvate carboxylase (PC) is required for breast to lung metastasis, and may play a role in promoting ECM detached cell survival by supporting metabolic plasticity. PC activity results in the carboxylation of pyruvate to synthesize the TCA cycle intermediate oxaloacetate (OAA). We hypothesize that PC expression is required for cell survival in ECM detached conditions. The Harvey-ras transformed MCF10A human breast epithelial cell line (MCF10A-ras) were utilized for these studies. ECM detachment was modeled using Poly-HEMA coated plates in combination with MTT assays to measure cell viability. qPCR was used to quantify mRNA abundance of metabolic genes. Protein expression was quantified using western blot. PC activity was determined using [13C6]-glucose flux analysis by comparing 3 carbon labeled (M+3) citrate, malate, and aspartate. Results show a 94% increase in PC mRNA expression and a 90% increase in PC protein expression, in detached cells compared to attached cells. In addition PC knockdown decreases viability in ECM detached conditions significantly by 15%, while PC overexpression increases viability by 17%. Further, replenishing the TCA cycle with OAA, the product of the PC reaction, rescues PC knockdown cells by 14%. Increased PC activity in detached cells was determined by a 23% increase in M+3 malate and a 61% increase in M+3 aspartate. However, an 11% decrease in M+3 citrate was observed, suggesting that detached cells do not utilize increased PC expression to replenish the TCA cycle intermediate, citrate. Given the larger relative increase in PC activity demonstrated by M+3 aspartate, we hypothesized that PC activity is required for detached cell survival to provide substrate for aspartate synthesis. Providing aspartate (2.5 mM), a downstream product of PC, in detached cells with PC knockdown rescued cell viability with no benefit of aspartate supplementation in detached PC expressing cells. Further, the aspartate synthesizing enzyme, glutamate OAA transferase 2 (GOT2) mRNA expression was unchanged in detached cells. In summary PC expression and activity is increased in detached cells compared to attached cells, and OAA produced by the PC reaction is utilized by cells to synthesize aspartate and maintain cell viability in detached conditions. PC may be a useful target for future treatments aimed at preventing metastasis. Citation Format: Madeline P. Sheeley, Dorothy Teegarden. Pyruvate carboxylase rescues MCF10A-ras cell viability in extracellular matrix detached conditions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2327.

Antibody\u2013drug nanoparticle induces synergistic treatment efficacies in HER2 positive breast cancer cells

Chemotherapeutic drugs suffer from non-specific binding, undesired toxicity, and poor blood circulation which contribute to poor therapeutic efficacy. In this study, antibody–drug nanoparticles (ADNs) are engineered by synthesizing pure anti-cancer drug nanorods (NRs) in the core of nanoparticles with a therapeutic monoclonal antibody, Trastuzumab on the surface of NRs for specific targeting and synergistic treatments of human epidermal growth factor receptor 2 (HER2) positive breast cancer cells. ADNs were designed by first synthesizing ~ 95 nm diameter × ~ 500 nm long paclitaxel (PTX) NRs using the nanoprecipitation method. The surface of PTXNRs was functionalized at 2′ OH nucleophilic site using carbonyldiimidazole and conjugated to TTZ through the lysine residue interaction forming PTXNR-TTZ conjugates (ADNs). The size, shape, and surface charge of ADNs were characterized using scanning electron microscopy (SEM), SEM, and zeta potential, respectively. Using fluorophore labeling and response surface analysis, the percentage conjugation efficiency was found > 95% with a PTX to TTZ mass ratio of 4 (molar ratio ≈ 682). In vitro therapeutic efficiency of PTXNR-TTZ was evaluated in two HER2 positive breast cancer cell lines: BT-474 and SK-BR-3, and a HER2 negative MDA-MB-231 breast cancer cell using MTT assay. PTXNR-TTZ inhibited > 80% of BT-474 and SK-BR-3 cells at a higher efficiency than individual PTX and TTZ treatments alone after 72 h. A combination index analysis indicated a synergistic combination of PTXNR-TTZ compared with the doses of single-drug treatment. Relatively lower cytotoxicity was observed in MCF-10A human breast epithelial cell control. The molecular mechanisms of PTXNR-TTZ were investigated using cell cycle and Western blot analyses. The cell cycle analysis showed PTXNR-TTZ arrested > 80% of BT-474 breast cancer cells in the G2/M phase, while > 70% of untreated cells were found in the G0/G1 phase indicating that G2/M arrest induced apoptosis. A similar percentage of G2/M arrested cells was found to induce caspase-dependent apoptosis in PTXNR-TTZ treated BT-474 cells as revealed using Western blot analysis. PTXNR-TTZ treated BT-474 cells showed ~ 1.3, 1.4, and 1.6-fold higher expressions of cleaved caspase-9, cytochrome C, and cleaved caspase-3, respectively than untreated cells, indicating up-regulation of caspase-dependent activation of apoptotic pathways. The PTXNR-TTZ ADN represents a novel nanoparticle design that holds promise for targeted and efficient anti-cancer therapy by selective targeting and cancer cell death via apoptosis and mitotic cell cycle arrest.

MicroRNA-181a suppresses norethisterone-promoted tumorigenesis of breast epithelial MCF10A cells through the PGRMC1/EGFR–PI3K/Akt/mTOR signaling pathway

BackgroundResearch suggests that hormone replacement therapy may increase the risk of breast cancer, and progestins such as norethisterone (NET) play a key role in this phenomenon. We have demonstrated that microRNA-181a (miR-181a) suppresses NET-promoted breast cancer cell survival. Nonetheless, the effects of NET and miR-181a on the tumorigenesis of human breast epithelial cells have not yet been elaborated. MethodsAssays of cell viability, proliferation, migration, apoptosis, and colony formation were performed to investigate the pro-tumorigenesis effect of NET and the effects of miR-181a on human breast epithelial MCF10A cells. The expressions of cell-proliferation-related genes and apoptotic factors were analyzed by quantitative RT-PCR and Western blot in MCF10A cells treated with NET and miR-181a. ResultsNET significantly increased MCF10A cell viability, proliferation, migration, and colony formation, but reduced cellular apoptosis. In addition, NET increased the expression of progesterone receptor membrane component 1 (PGRMC1), EGFR, B-cell lymphoma 2, cyclin D1, and proliferating cell nuclear antigen, but decreased the expression of pro-apoptosis factors, such as Bax, caspase-7, and caspase-9. Overexpression of miR-181a strongly inhibited the effects of NET on MCF10A cells and abrogated NET-stimulated PGRMC1, EGFR, and mTOR expression. ConclusionsActivation of the PGRMC1/EGFR–PI3K/Akt/mTOR signaling pathway is the primary mechanism underlying the pro-tumorigenesis effects of NET on human breast epithelial MCF10A cells. Additionally, miR-181a can suppress the effects of NET on these cells. These data suggest a therapeutic potential for miR-181a in reducing or preventing the risk of breast cancer in hormone replacement therapy using NET.

Licochalcone A is a Natural SelectiveInhibitor of Arginine Methyltransferase 6.

Arginine methylation is a post-translational modification that is implicated in multiple biological functions including transcriptional regulation. The expression of protein arginine methyltransferases (PRMT) has been shown to be up-regulated in various cancers. PRMTs have emerged as attractive targets for the development of new cancer therapies. Here, we describe the identification of a natural compound, licochalcone A, as a novel, reversible and selective inhibitor of PRMT6. Since expression of PRMT6 is up-regulated in human breast cancers and is associated with oncogenesis, we used the human breast cancer cell line system to study the effect of licochalcone A treatment on PRMT6 activity, cell viability, cell cycle, and apoptosis. We demonstrated that licochalcone A is a non-S-adenosyl L-methionine (SAM) binding site competitive inhibitor of PRMT6. In MCF-7 cells, it inhibited PRMT6-dependent methylation of histone H3 at arginine 2 (H3R2), which resulted in a significant repression of estrogen receptor activity. Licochalcone A exhibited cytotoxicity towards human MCF-7 breast cancer cells, but not MCF-10A human breast epithelial cells, by up-regulating p53 expression and blocking cell cycle progression at G2/M, followed by apoptosis. Thus, licochalcone A has potential for further development as a therapeutic agent against breast cancer.

Cytotoxicity and genotoxicity of Hevea brasiliensis latex C-serum DCS sub-fraction as anticancer agents

Dialysed latex C-serum supernatant (DCS) sub-fraction from the rubber tree (Hevea brasiliensis) was reported to exert significant anti-proliferative effect on specific cancer cell lines, especially in human triple negative breast cancer (MDA-MB-231) and human liver carcinoma (HepG2) cells. In the present study, two human non-cancer origin cell lines, MCF-10A (human breast epithelial cells) and HDFa (human adult dermal fibroblast cells), showed least susceptibility when treated with DCS sub-fraction at concentration range 0–100 µg/mL using 2,5-diphenyl tetrazolium bromide (MTT) assay. Genotoxicity of DCS sub-fraction was assessed through Ames test, in vitro mouse lymphoma assay, and in vitro micronucleus assay. All genotoxicity experiments were tested with the presence and absence of S9 mixture as metabolic activation system. Ames test results showed negative result of mutagenicity as no clear dose-dependent relationship was observed despite mutagenic potential was detected in frame shift-based TA98 and TA1535 Salmonella typhimurium strains. Mouse lymphoma assay produced non-linear dose-dependent effect in experiments without the presence of S9. Meanwhile, experiment with the presence of S9 resulted in an inverse effect of the dose-dependent relationship. In micronucleus assay, the dose-dependent cytotoxicity effect of DCS sub-fraction showed no correlation with the increase number of cells containing micronuclei but appeared to be time-dependent. In conclusion, DCS sub-fraction showed no significant positive results in genotoxicity assays. In vivo genotoxicity assays should be conducted to ensure the safe use of DCS sub-fraction in further exploration for biological applications.

Abstract 2571: 1,25-dihydroxyvitamin D regulation of pyruvate carboxylase in MCF10A-ras cells decreases viability in extracellular matrix detached conditions

Abstract Metastasis is the main cause of most breast cancer related deaths. Throughout the process of metastasis, cancer cells strive to survive in conditions where they are detached from the extracellular matrix (ECM). Vitamin D may play a role in preventing breast cancer metastasis by modulating metabolism in breast cancer cells detached from ECM. The anaplerotic enzyme pyruvate carboxylase (PC) is essential for breast to lung metastasis and the active metabolite of vitamin D, 1,25-dihydroxyvitamin D3 (1,25D), downregulates PC expression through a vitamin D response element. PC replenishes the TCA cycle with the intermediate oxaloacetate (OAA), and lower expression of PC following 1,25D treatment may cause impaired energy metabolism via the TCA cycle. We hypothesize that the 1,25D mediated decrease in PC expression decreases breast cancer cell viability in ECM detached conditions. The Harvey-ras transformed MCF10A human breast epithelial cell line (MCF10A-ras) treated with vehicle (ethanol) or 10 nM 1,25D and maintained in varying nutrient concentrations were utilized for these studies. Poly-HEMA coated plates were used to model ECM detachment paired with MTT assays to measure cell viability. mRNA expression of metabolic genes is quantified using qPCR. Results show that when compared to attached cells, detached cells have a 94% increase in PC mRNA expression, while 1,25D treatment decreases PC mRNA expression in both attached by 75% and detached conditions by 63%. In addition 1,25D treatment and PC knockdown decreases viability in ECM detached conditions significantly by 16% and 11%, with no additional effect when combined. Glutamine deprivation also decreases viability by 40% in detached conditions, however, the combination of 1,25D treatment and glutamine deprivation results in a 71% decrease in viability. These results suggest that while glutamine is required for ECM survival, regulation of glutamine metabolism is not the mechanism by which 1,25D affects ECM survival. Replenishing the TCA cycle with OAA rescues glutamine depleted cells by 84% as well as PC knockdown cells by 14%. Further, when stressed with low glutamine conditions, cells in attached conditions increase mRNA expression of PC by 311%, suggesting that changes in PC expression may be a mechanism that cancer cells use to adapt to energy related stress. These results suggest that 1,25D regulation of metabolism may sensitize breast cancer cells to ECM detachment death via downregulation of PC, reducing the required flux of substrates into the TCA cycle which are needed to survive ECM detached conditions during metastasis. Citation Format: Madeline Sheeley, Dorothy Teegarden, Katie Wong. 1,25-dihydroxyvitamin D regulation of pyruvate carboxylase in MCF10A-ras cells decreases viability in extracellular matrix detached conditions [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2571.

Evaluation of the Anticancer Activities of Novel Transition Metal Complexes with Berenil and Nitroimidazole.

Novel transition metal complexes (Au, Pd, Pt) with berenil and 2-(1-methyl-5-nitroimidazol-2-yl)ethanol were obtained through two-step synthesis. The cytotoxicity assay against MCF-7 and MDA-MB-231 breast cancer cells revealed that novel platinum and palladium complexes cause a reduction on the viability of MCF-7 and MDA-MB-231 breast cancer cells to a greater extent than cisplatin. The complexes showed lower cytotoxicity on normal MCF-10A human breast epithelial cells than on tumor cells. Furthermore, we observed that these complexes selectively concentrate in tumor cell mitochondria due to the characteristic for these cells increased membrane potential that may explain their increased proapoptotic activity. The activity of the synthesized compounds against topoisomerase type IIα and their increased impact on DNA defragmentation also were documented. The novel complexes also induced autophagosome changes and inhibited tumor growth in xenograft models (established using breast cancer cells).

SAT-136 Hypoxia Effect on Cytokine Production by Breast Cancer Cells

Inflammation is a critical component of tumor initiation and progression. Chronic inflammation triggers molecular events that can promote carcinogenesis, tumor vascularization and metastasis. As inflammatory mediators, cytokines play an important role in the interplay between the tumor cells and tumor microenvironment. Cytokines released by the tumor-associated macrophages modulate cancer cell survival, stemness, invasiveness, and tumor vascularization. Breast cancer cells, however, also produce a variety of cytokines, whose role in cancer development is poorly understood. The aim of our study was to characterize the basal cytokine secretory activity in commonly used human breast cancer cell lines (MDA-MB-231, MCF-7, BT-474, and T-47D). Using MILLIPLEX assay, we measured the expression of 41 cytokines, including interleukins, monokines, interferons and growth factors. We also compared cytokine expression profile of breast cancer cells with those of non-tumorigenic human breast epithelial MCF-10A cells. Further, we investigated whether hypoxia modulates cytokine secretion of breast cancer cells. Using cobalt(II) chloride (CoCl2) to mimic hypoxia, we compared the effect of various treatment doses and intervals on cytokine production in the breast cancer cells. Results demonstrated that CoCl2 affects the release of multiple cytokines in a dose- and concentration-dependent manner, thus highlighting the role of cancer cell-derived cytokines on breast tumor progression. Understanding the molecular actions of cytokines in the tumor microenvironment is important for understanding the mechanism of cancer initiation and progression.

Roles of C‐C motif chemokine ligand (CCL) 2 in aggressive phenotypic change of non‐neoplastic breast cells by tumor‐associated macrophages

Tumor‐associated macrophages (TAMs) are major components of tumor microenvironment that promote invasion and metastasis of cancer cells. In this study, we investigated the effect of TAMs on phenotypic conversion of non‐neoplastic MCF10A human breast epithelial cells using an indirect co‐culture system. Co‐culture with TAMs induced epithelial‐to‐mesenchymal transition, invasive phenotype, and MMP‐9 upregulation in MCF10A cells. Comparative proteomic analysis revealed that endoplasmic reticulum oxidoreductase (ERO)1‐α was increased in MCF10A cells co‐cultured with TAMs compared to that in mono‐cultured cells. ERO1‐α was crucial for TAMs‐induced invasive phenotype and MMP‐9 upregulation involving transcription factors c‐fos and c‐Jun. Cytokine array analysis showed that levels of interleukin (IL)‐6, C‐X‐C motif ligand (CXCL)1, C‐C motif ligand (CCL)2, growth‐regulated oncogene (GRO), IL‐8, and granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) were increased in conditioned media of co‐cultured cells. Among these cytokines increased in conditioned media of co‐cultured cells, CCL2 was secreted from TAMs, leading to induction of ERO1‐α, MMP‐9 upregulation, and invasiveness in MCF10A cells. Our findings elucidated a molecular mechanism underlying the aggressive phenotypic change of non‐neoplastic breast cells by co‐culture with TAMs, providing useful information for prevention or treatment of recurrent breast cancer.Support or Funding InformationThe present study was supported by the National Research Foundation of Korea (No. 2013R1A2A2A04013379, No.2015M3A9B6074045, and No. 2016R1A6A1A03007648).

Statin-induced GGPP depletion blocks macropinocytosis and starves cells with oncogenic defects

Cancer cells display novel characteristics which can be exploited for therapeutic advantage. Isolated studies have shown that 1) the mevalonate pathway and 2) increased macropinocytosis are important in tumorigenesis, but a connection between these two observations has not been envisioned. A library screen for compounds that selectively killed Dictyostelium pten- cells identified pitavastatin. Pitavastatin also killed human breast epithelial MCF10A cells lacking PTEN or expressing K-RasG12V, as well as mouse tumor organoids. The selective killing of cells with oncogenic defects was traced to GGPP (geranylgeranyl diphosphate) depletion. Disruption of GGPP synthase in Dictyostelium revealed that GGPP is needed for pseudopod extension and macropinocytosis. Fluid-phase uptake through macropinocytosis is lower in PTEN-deleted cells and, as reported previously, higher in cells expressing activated Ras. Nevertheless, uptake was more sensitive to pitavastatin in cells with either of these oncogenic mutations than in wild-type cells. Loading the residual macropinosomes after pitavastatin with high concentrations of protein mitigated the cell death, indicating that defective macropinocytosis leads to amino acid starvation. Our studies suggest that the dependence of cancer cells on the mevalonate pathway is due to the role of GGPP in macropinocytosis and the reliance of these cells on macropinocytosis for nutrient uptake. Thus, inhibition of the networks mediating these processes is likely to be effective in cancer intervention.

Integration of proteomics and metabolomics reveals promotion of proliferation by exposure of bisphenol S in human breast epithelial MCF-10A cells

Bisphenol S (BPS) has been reported to have similar estrogenic effects as bisphenol A (BPA). Considering the endocrine disrupting effects of BPS, in this study, we investigated the effects of BPS exposure on normal human breast epithelial cell line MCF-10A by using mass spectrometry (MS)-based metabolomics and quantitative proteomics. We found that exposure to BPS for 24 h altered the proliferation of MCF-10A cells in a hormetic manner with the highest proliferation rate at the dosage of 1 μM. A total of 200 proteins were identified to be significantly changed by 1 μM of BPS exposure. The upregulation of epidermal growth factor receptor (EGFR) and Ras/mTOR-related proteins implied that EGFR-mediated pathways were involved in BPS-induced proliferation of MCF-10A cells. In addition, several proliferation-related protein markers were found to be elevated, such as MKI67 and CDH1, further indicating the promotion of proliferation by low dose of BPS exposure. Besides, 35 endogenous metabolites were found to be significantly changed. The joint pathway analysis of the altered metabolites and proteins suggested changes in pathways of tricarboxylic acid (TCA) cycle, purine metabolism, pyruvate metabolism and lipid metabolism, which were involved in sustaining cell proliferation and cellular signal transduction. Taken together, this study provides insights into the effects and the potential mechanisms of BPS on estrogen receptor α-negative normal breast cell line MCF-10A, broadening our knowledge about the risk of using BPS as the alternative of BPA.

Zn(ii), Cd(ii) and Hg(ii) saccharinate complexes with 2,6-bis(2-benzimidazolyl)pyridine as promising anticancer agents in breast and lung cancer cell lines via ROS-induced apoptosis.

New Zn(ii), Cd(ii) and Hg(ii) complexes of saccharinate (sac) and 2,6-bis(2-benzimidazolyl)pyridine (bzimpy), [Zn(bzimpy)2](sac)2·2H2O (Zn), [Cd(sac)2(bzimpy)] (Cd) and [Hg(sac)2(bzimpy)] (Hg), were prepared and fully characterized by spectroscopic methods and X-ray crystallography. In vitro anticancer screening in A549 (lung), MCF-7 (breast) and HT29 (colon) cell lines showed that Zn was highly cytotoxic against A549 and MCF-7 cells with IC50 values of 1.74 ± 0.06 and 3.15 ± 0.10 μM, respectively, and Hg demonstrated potent cytotoxic activity in MCF-7 cells (8.61 ± 0.98 μM), while Cd and bzimpy exhibited moderate growth inhibitory activities in all of the cell lines. In addition, they showed significantly lower toxicity towards normal human breast epithelial MCF10A cells. Moreover, the complexes exhibited significantly high nuclease activity towards plasmid DNA and their interactions with DNA were assessed by gel electrophoresis and DNA docking. Zn and Hg induced G0/G1 cell arrest and apoptotic cell death detected via typical DNA condensation/fragmentation, annexin V staining and caspase 3/7 activity in A549 and MCF-7 cells. These complexes further caused depolarization of mitochondria and oxidative damage of genomic DNA following excessive production of reactive oxygen species (ROS).

Abstract 129: Tumor-associated macrophages secrete CCL2 and induce the invasive phenotype of human breast epithelial cells through upregulation of ERO1-alpha and MMP-9

Abstract Tumor-associated macrophages (TAMs) are major components of tumor microenvironment that promote invasion and metastasis of cancer cells. In this study, we investigated the effect of TAMs on phenotypic conversion of non-neoplastic MCF10A human breast epithelial cells using an indirect co-culture system. Co-culture with TAMs induced epithelial-to-mesenchymal transition, invasive phenotype, and MMP-9 upregulation in MCF10A cells. Comparative proteomic analysis revealed that endoplasmic reticulum oxidoreductase (ERO)1-alpha was increased in MCF10A cells co-cultured with TAMs compared to that in mono-cultured cells. ERO1-alpha was crucial for TAMs-induced invasive phenotype and MMP-9 upregulation involving transcription factors c-fos and c-Jun. Cytokine array analysis showed that levels of interleukin (IL)-6, C-X-C motif ligand (CXCL)1, C-C motif ligand (CCL)2, growth-regulated protein (GRO), IL-8, and granulocyte-macrophage colony-stimulating factor (GM-CSF) were increased in conditioned media of co-cultured cells. Among these cytokines increased in conditioned media of co-cultured cells, CCL2 was secreted from TAMs, leading to induction of ERO1-alpha, MMP-9 upregulation, and invasiveness in MCF10A cells. Our findings elucidated a molecular mechanism underlying the aggressive phenotypic change of non-neoplastic breast cells by co-culture with TAMs, providing useful information for prevention or treatment of recurrent breast cancer Note: This abstract was not presented at the meeting. Citation Format: Seungeun Lee, Eunhye Lee, EunYi Ko, Mina Ham, Hye Min Lee, Eun-Sook Kim, Minsoo Koh, Hyun Kyung Lim, Joohee Jung, So Yeon Park, Aree Moon. Tumor-associated macrophages secrete CCL2 and induce the invasive phenotype of human breast epithelial cells through upregulation of ERO1-alpha and MMP-9 [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 129.

1, 25-Dihydroxyvitamin D3 Altered Metabolism of MCF10A-ras Cells Decreases Viability in Extracellular Matrix Detached Conditions (FS13-05-19)

ObjectivesMetastasis is the main cause of most breast cancer related deaths. Throughout the process of metastasis, cancer cells strive to survive in conditions where they are detached from the extracellular matrix (ECM). Vitamin D may play a role in preventing breast cancer metastasis by modulating metabolism in breast cancer cells detached from ECM. Here we hypothesize that the active metabolite of vitamin D, 1,25-dihydroxyvitamin D3(1,25D), regulates the metabolism of breast cancer cells, and decreases their viability in ECM detached conditions. MethodsThe Harvey-ras transformed MCF10A human breast epithelial cell line (MCF10A-ras) were utilized for these studies. Poly-HEMA coated plates were used to model ECM detachment paired with MTT assays to measure cell viability. Viability is measured in cells treated with vehicle (ethanol) or 10 nM 1,25D and maintained in varying nutrient concentrations. mRNA expression of metabolic genes is quantified using qPCR and, triacylglycerol (TAG) is quantified using a Wako Diagnostics colorimetric assay. ResultsResults show that 1,25D treatment decreases viability in detached conditions by 23%. Glutamine deprivation also decreases viability in detached conditions by 40%. In addition, in combination 1,25D treatment and glutamine deprivation results in a 71% decrease in viability. These results imply that while glutamine is required for ECM survival, 1,25D’s modulation of metabolism is targeted elsewhere. Further, when compared to vehicle, 1,25D treated cells have a 45% increased TAG accumulation prior to plating in detached conditions. A 50% decrease was observed in mRNA expression of the enzyme pyruvate carboxylase (PC), and replenishing the product of PC and tricarboxylic acid (TCA) cycle intermediate oxaloacetate rescues detached cell viability in glutamine deprived conditions by 84%. ConclusionsThese results demonstrate the important role energy metabolism plays in cell survival during ECM detachment. Furthermore, our results suggest that 1,25D regulation of metabolism may sensitize breast cancer cells to ECM detachment death. We conclude that cells detached from ECM require flux into the TCA cycle in order to survive, and 1,25D’s downregulation of PC contributes to the observed decrease in viability following 1,25D treatment. Funding SourcesNational Institute of Health and USDA.

Crude Edible Fig (Ficus carica) Leaf Extract Prevents Diethylstilbestrol (DES)-Induced DNA Strand Breaks in Single-Cell Gel Electrophoresis (SCGE)/Comet Assay: Literature Review and Pilot Study.

Fig (Ficus carica) trees are among the oldest plants on earth. The chemopreventive properties of constituent polyphenols and fiber that implicate figs in having a functional role in averting cancer have not been fully elucidated. We therefore hypothesized that fig leaf extract would inhibit (or attenuate) DES-induced DNA single-strand breakage in MCF10A human breast epithelial cells. To test this hypothesis, MCF10A cells were treated with DES (1, 10, 100 μM), crude fig leaf extract (5, 10, 15 μL), or concomitant doses of DES (100 μM)/fig leaf extract (5, 10, 15 μL). The cells were analyzed for DNA strand breakage using the SCGE/COMET assay with mean olive tail moment as a marker of DNA damage. DES induced DNA strand breaks at all treatment levels compared to DMSO and non-treatment controls. DES at concentrations of 1, 10, and 100 μM produced mean olive tail moments of 1.2082 (177.6%), 1.2702 (186.7%), and 1.1275 (165.7%), respectively, which were statistically significantly (p<0.05) higher than the DMSO control value (0.6803). Exposure to fig leaf extract produced no DNA damage. Rather, a desirable dose-dependent reduction in DES-induced DNA strand breaks was observed. Composite treatment of MCF10A cells with DES and fig leaf extract attenuated DES-induced DNA strand breaks. Taken together, these results suggest a potential mechanism for cancer chemoprevention. Additional studies are necessary to identify relevant active ingredients, confirm the mechanism of action, and further elucidate the therapeutic potential of fig leaf extract for early-stage breast cancer chemoprevention.

MiR-1207-5p regulates the sensitivity of triple-negative breast cancer cells to Taxol treatment via the suppression of LZTS1 expression.

Taxol-based chemotherapy is a conventional therapeutic approach for the treatment of triple-negative breast cancer (TNBC). However, the insensitivity of TNBC cells to Taxol greatly limits the anticancer effect of the drug and leads to patient mortality. The present study first evaluated the expression levels of microRNA (miR)-1207-5p in human normal breast epithelial MCF-10A cells and TNBC cell lines (MDA-MB-231, MDA-MB-436 and MDA-MB-453). The results revealed that the highest miR-1207-5p level was in MDA-MB-231, which suggested an oncogenic role of miR-1207-5p in TNBC. Therefore, MDA-MB-231 served as the present study's research model in subsequent experiments. The mRNAs that functioned as tumor suppressor factors for miR-1207-5p were then determined. Leucine zipper tumor suppressor gene 1 (LZTS1), which was predicted by TargetScan 6.2 and was supported by the results of a dual luciferase assay, was identified as a target of miR-1207-5p. AntagomiR-1207-5p increased LZTS1 mRNA and protein expressions, enhanced cell growth arrest and cell apoptosis induced by Taxol in MDA-MB-231 cells. Additionally, it was observed that, when compared with Taxol treatment, the combination of Taxol and antagomiR-1207-5p induced a sharp decrease in B-cell lymphoma 2 (Bcl-2) and phosphorylated-protein kinase B expression accompanied by an increase in the Bcl-2-associated X protein expression. Finally, miR-1207-5p expression was significantly increased, while LZTS1 expression was significantly decreased, in TNBC tissues when compared with normal adjacent tissues, and there was a negative correlation between miR-1207-5p and LZTS1 expression. In addition, there was a notable elevation in the expression of miR-1207-5p and a reduction in the expression of LZTS1 in the Taxol non-responsive TNBC tissues when compared with the Taxol-responsive TNBC tissues. The results of the present study suggested that miR-1207-5p may be a promising predictor of sensitivity towards Taxol in TNBC.

Tumor-associated macrophages secrete CCL2 and induce the invasive phenotype of human breast epithelial cells through upregulation of ERO1-α and MMP-9

Tumor-associated macrophages (TAMs) are major components of tumor microenvironment that promote invasion and metastasis of cancer cells. In this study, we investigated the effect of TAMs on phenotypic conversion of non-neoplastic MCF10A human breast epithelial cells using an indirect co-culture system. Co-culture with TAMs induced epithelial-to-mesenchymal transition, invasive phenotype, and MMP-9 upregulation in MCF10A cells. Comparative proteomic analysis revealed that endoplasmic reticulum oxidoreductase (ERO)1-α was increased in MCF10A cells co-cultured with TAMs compared to that in mono-cultured cells. ERO1-α was crucial for TAMs-induced invasive phenotype and MMP-9 upregulation involving transcription factors c-fos and c-Jun. Cytokine array analysis showed that levels of interleukin (IL)-6, C-X-C motif ligand (CXCL)1, C-C motif ligand (CCL)2, growth-regulated protein (GRO), IL-8, and granulocyte-macrophage colony-stimulating factor (GM-CSF) were increased in conditioned media of co-cultured cells. Among these cytokines increased in conditioned media of co-cultured cells, CCL2 was secreted from TAMs, leading to induction of ERO1-α, MMP-9 upregulation, and invasiveness in MCF10A cells. Our findings elucidated a molecular mechanism underlying the aggressive phenotypic change of non-neoplastic breast cells by co-culture with TAMs, providing useful information for prevention or treatment of recurrent breast cancer.

Abstract B27: Transfection of mutant P53 promotes membrane translocation of GLUT1 and increases glucose uptake in MCF10A human breast epithelial cells

Abstract Background: High rate of glycolysis exhibited by cancer cells plays a crucial role in carcinogenesis and tumor progression. Previous study showed that gain-of-function (GOF) by mutant P53 drives the Warburg effect via GLUT1 membrane translocation in malignancies. Also, in the patients with a preoperative 18F-fluorodeoxy glucose positron emission tomography (FDG-PET), which enables to estimate glucose uptake by tumor as standardized uptake value (SUV), we identified high SUV as a poor prognostic marker. In this study, we wondered whether P53 promotes GLUT1 membrane translocation and increases glucose uptake in human normal breast epithelial cells using MCF10A cells. Methods: We generated mutant-P53 MCF10A cell line, which include sequence variants (R175H, R273H, and R248W). First, to generate stable P53 knockdown cell lines, lentiviral transduction particles from TRC-pLKO-U6 shP53 were infected into MCF10A. To generate mutant P53 producing MCF10A, pcDNA mutant P53 was transfected using lipofectamin (Invitrogen) into stable P53 knockdown cell lines. Glucose uptake assay and confocal imaging studies with 2NDBG were performed. Confocal imaging system was used to detect GLUT1 membrane expression. In case-matched patients according to P53 mutation (n=114), we compared SUV. Mutational analysis of exons 5-9 of the P53 gene was carried out using Sanger sequencing. Results: Glucose uptake assay showed that the level of glucose uptake was increased in P53 mutant MCF10A cell lines (R175H or R273H, and R248W) with a 1.5-1.8 fold change compared with wild-type MCF10A. The increase of glucose uptake by mutant-P53 was reproducible with the experiments with 2NBGD. The mRNA expressional level of LDHA was also upregulated by mutant P53. In contrast, the mRNA expression of GLUT1, hexokinase 2, PKM1, PKM2, and G6PD was not elevated by P53 mutation. The confocal imaging studies captured translocation to membrane of GLUT1 in mutant P53-MCF10A. However, in wild-type MCF10A, GLUT1 was diffusely expressed in cytoplasm. In patients, the mean SUV of p53-mutational group was significantly higher than that of wild-type p53-group (7.49 vs. 5.44, P=0.013). Compared with the group with intact p53, recurrence-free survival (RFS) was significantly reduced in the group with p53 mutation (P =0.017). In multivariate analysis, p53 mutational status carried prognostic significance (hazard ratio 3.73, 95% CI 1.15-12.07) independent of tumor size, nodal status, and estrogen receptor status. Conclusion: We showed that P53 mutation promotes GLUT1 translocation to membrane, which consequently induces glucose influx in MCF10A cells, elucidating that P53 mutation contributes to the increase of SUV in patients with breast cancer. Citation Format: Sung Gwe . Ahn, Hyekyoung Shin, Bae Soong June, Yoon Changik, Lee Hak Woo, Jeong Joon. Transfection of mutant P53 promotes membrane translocation of GLUT1 and increases glucose uptake in MCF10A human breast epithelial cells [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr B27.

Effects of PTEN Loss and Activated KRAS Overexpression on Mechanical Properties of Breast Epithelial Cells.

It has previously been shown that the simultaneous activation of PI3K (phosphatidylinositol 3-kinase) and Ras/MAPK (mitogen-activated protein kinases) pathways facilitate tumor growth despite only inducing cancer cell dormancy individually. Determining the impacts on cellular mechanics each pathway incites alone and in unison is critical to developing non-toxic cancer therapies for triple-negative breast cancers. PTEN (phosphatase and tensin homolog) knockout and activated KRAS (Kristen rat sarcoma viral oncogene homolog) overexpression in healthy MCF-10A human breast epithelial cells activated the PI3K and Ras/MAPK pathways, respectively. Cell stiffness and fluidity were simultaneously measured using atomic force microscopy. Results suggest that PTEN knockout reduced cell stiffness and increased cell fluidity independent of PI3K activation. Effects of activated KRAS overexpression on cell stiffness depends on rigidity of cell culture substrate. Activated KRAS overexpression also counteracts the effects of PTEN knockout.