Non-tumorigenic Human Breast Epithelial Cells Research Articles

Interfacial Organization and Forces Arising from Epithelial-Cancerous Monolayer Interactions.

The interfacial interactions between epithelia and cancer cells have profound relevance for tumor development and metastasis. Through monolayer confrontation of MCF10A (nontumorigenic human breast epithelial cells) and MDA-MB-231 (human epithelial breast cancer cells) cells, we investigate the epithelial-cancerous interfacial interactions at the tissue level. We show that the monolayer interaction leads to competitive interfacial morphodynamics and drives an intricate spatial organization of MCF10A cells into multicellular finger-like structures, which further branch into multiple subfinger-like structures. These hierarchical interfacial structures penetrate the cancer monolayer and can spontaneously segregate or even envelop cancer cell clusters, consistent with our theoretical prediction. By tracking the substrate displacements via embedded fluorescent nanobeads and implementing nanomechanical modeling that combines atomic force microscopy and finite element simulations, we computed mechanical force patterns, including traction forces and monolayer stresses, caused by the monolayer interaction. It is found that the heterogeneous mechanical forces accumulated in the monolayers are able to squeeze cancer cells, leading to three-dimensional interfacial bulges or cell extrusion, initiating the p53 apoptosis signaling pathways of cancer cells. We reveal that intercellular E-cadherin and P-cadherin of epithelial cells differentially regulate the interfacial organization including migration speed, directionality, spatial correlation, F-actin alignment, and subcellular protrusions of MCF10A cells; whereas E-cadherin governs interfacial geometry that is relevant to force localization and cancer cell extrusion, P-cadherin maintains interfacial integrity that enables long-range force transmission. Our findings suggest that the collaborative molecular and mechanical behaviors are crucial for preventing epithelial tissues from undergoing tumor invasion.

Adipocyte-derived kynurenine stimulates malignant transformation of mammary epithelial cells through the aryl hydrocarbon receptor

Anti-hormone therapies are not efficacious for reducing the incidence of triple negative breast cancer (TNBC), which lacks both estrogen and progesterone receptors. While the etiology of this aggressive breast cancer subtype is unclear, visceral obesity is a strong risk factor for both pre- and post-menopausal cases. The mechanism by which excessive deposition of visceral adipose tissue (VAT) promotes the malignant transformation of hormone receptor-negative mammary epithelial cells is currently unknown. We developed a novel in vitro system of malignant transformation in which non-tumorigenic human breast epithelial cells (MCF-10A) grow in soft agar when cultured with factors released from VAT. These cells, which acquire the capacity for 3D growth, show elevated aryl hydrocarbon receptor (AhR) protein and AhR target genes, suggesting that AhR activity may drive malignant transformation by VAT. AhR is a ligand-dependent transcription factor that generates biological responses to exogenous carcinogens and to the endogenous tryptophan pathway metabolite, kynurenine. The serum kynurenine to tryptophan ratio has been shown to be elevated in patients with obesity. Herein, we demonstrate that AhR inhibitors or knockdown of AhR in MCF-10A cells prevents VAT-induced malignant transformation. Specifically, VAT-induced transformation is inhibited by Kyn-101, an inhibitor for the endogenous ligand binding site of AhR. Mass spectrometry analysis demonstrates that adipocytes metabolize tryptophan and release kynurenine, which is taken up by MCF-10A cells and activates the AhR to induce CYP1B1 and promote malignant transformation. This novel hormone receptor-independent mechanism of malignant transformation suggests targeting AhR for TNBC prevention in the context of visceral adiposity.

Theoretical, in Vitro Antiproliferative, and in Silico Molecular Docking and Pharmacokinetics Studies of Heteroleptic Nickel(II) and Copper(II) Complexes of Thiosemicarbazone-Based Ligands and Pefloxacin.

Twelve new heteroleptic nickel(II) and copper(II) complexes of the type [M(L1-6 )(Pfx)2 ] (1-12), where L1-6 =2-benzylidenehydrazinecarbothioamide (L1 ), 2-benzylidene-N-methylhydrazinecarbothioamide (L2 ), 2-benzylidene-N-phenylhydrazinecarbothioamide (L3 ), 2-(4-methylbenzylidene)hydrazinecarbothioamide (L4 ), 2-(4-methylbenzylidene)-N-methylhydrazinecarbothioamide (L5 ) and 2-(4-methylbenzylidene)-N-phenylhydrazinecarbothioamide (L6 ), Pfx=pefloxacin and M=Ni(II) or Cu(II) have been synthesised, and their structures were confirmed by different spectral techniques. The spectral data and density functional theory (DFT) calculations supported the bonding of pefloxacin drug molecule via one of the carboxylate oxygen atoms and the pyridone oxygen atom, and the thiosemicarbazone ligand via the imine nitrogen and the thione sulfur atoms with the metal(II) ion, forming distorted octahedral geometry. In vitro antiproliferative activity of the synthesized complexes was evaluated against three human breast cancer (T47D, estrogen negative (MDA-MB-231) and estrogen positive (MCF-7)) as well as non-tumorigenic human breast epithelial (MCF-10a) cell lines, which showed the higher activity for the copper(II) complexes. The interaction of the synthesized complexes with an oncogenic protein H-ras (121 p) was explored by in silico molecular docking studies. Further, in silico pharmacokinetics and ADMET parameters were also analysed to predict the drug-likeness as well as non-toxic and non-carcinogenic behavior, and safe oral administration of the complexes.

SNP rs4971059 predisposes to breast carcinogenesis and chemoresistance via TRIM46-mediated HDAC1 degradation.

Identification of the driving force behind malignant transformation holds the promise to combat the relapse and therapeutic resistance of cancer. We report here that the single nucleotide polymorphism (SNP) rs4971059, one of 65 new breast cancer risk loci identified in a recent genome-wide association study (GWAS), functions as an active enhancer of TRIM46 expression. Recreating the G-to-A polymorphic switch caused by the SNP via CRISPR/Cas9-mediated homologous recombination leads to an overt upregulation of TRIM46. We find that TRIM46 is a ubiquitin ligase that targets histone deacetylase HDAC1 for ubiquitination and degradation and that the TRIM46-HDAC1 axis regulates a panel of genes, including ones critically involved in DNA replication and repair. Consequently, TRIM46 promotes breast cancer cell proliferation and chemoresistance invitro and accelerates tumor growth invivo. Moreover, TRIM46 is frequently overexpressed in breast carcinomas, and its expression is correlated with lower HDAC1 expression, higher histological grades, and worse prognosis of the patients. Together, our study links SNP rs4971059 to replication and to breast carcinogenesis and chemoresistance and support thepursuit of TRIM46 as a potential target for breast cancer intervention.

Antitumor Effects of a Sesquiterpene Derivative from Marine Sponge in Human Breast Cancer Cells.

In this study, the anti-proliferative effect of ilimaquinone, a sesquiterpene derivative from the marine sponge, in breast cancer cells was investigated. Ilimaquinone inhibited the proliferation of MCF-7 and MDA-MB-231 breast cancer cells with IC50 values of 10.6 μM and 13.5 μM, respectively. Non-tumorigenic human breast epithelial cells were less sensitive to ilimaquinone than breast cancer cells. Flow cytometric and Western blot analysis showed that ilimaquinone induced S-phase arrest by modulating the expression of p-CDC-2 and p21. Ilimaquinone induces apoptosis, which is accompanied by multiple biological biomarkers, including the downregulation of Akt, ERK, and Bax, upregulation of p38, loss of mitochondrial membrane potential, increased reactive oxygen species generation, and induced autophagy. Collectively, these findings suggest that ilimaquinone causes cell cycle arrest as well as induces apoptosis and autophagy in breast cancer cells.

Facile synthesis of C6-substituted benz[4,5]imidazo[1,2-a]quinoxaline derivatives and their anticancer evaluation.

Cancer remains a leading cause of death worldwide, resulting in continuous efforts to discover and develop highly efficacious anticancer drugs. High-throughput screening of heterocyclic compound libraries is one of the promising approaches that provided several new lead molecules with a novel mechanism of action. On the basis of the promising anticancer potential of imidazoquinoxaline as well as the structurally similar imidazoquinoline-derived scaffold, we prepared a set of C6-substituted benzimidazo[1,2-a]quinoxaline derivatives via two novel synthetic routes using commercially available starting materials, with good to excellent yields and evaluated their anticancer activity against the NCI-60 cancer cell lines. The one-dose (10 µM) anticancer screening of the synthesized compounds in the NCI-60 cell line panel revealed that the substituents have a significant role in the activity. In particular, the indole (7f), imidazole (7g),and benzimidazole (7h) derivatives showed significant activity against the triple-negative breast cancer cell line, MDA-MB-468. The lead compounds also exhibited notable IC50 values against another breast cancer cell line, MCF-7. Furthermore, it was observed that these compounds were relatively nontoxic to normal cell lines: HEK293 (human embryonic kidney cell line) and MCF12A (nontumorigenic human breast epithelial cell line). The IC50 values against healthy cells were at least 5- to 11-fold higher, offering a new class of heterocycles that can be further developed as promising therapeutics for cancer treatment.

Signs of carcinogenicity induced by parathion, malathion, and estrogen in human breast epithelial cells (Review).

Cancer development is a multistep process that may be induced by a variety of compounds. Environmental substances, such as pesticides, have been associated with different human diseases. Organophosphorus pesticides (OPs) are among the most commonly used insecticides. Despite the fact that organophosphorus has been associated with an increased risk of cancer, particularly hormone-mediated cancer, few prospective studies have examined the use of individual insecticides. Reported results have demonstrated that OPs and estrogen induce a cascade of events indicative of the transformation of human breast epithelial cells. In vitro studies analyzing an immortalized non-tumorigenic human breast epithelial cell line may provide us with an approach to analyzing cell transformation under the effects of OPs in the presence of estrogen. The results suggested hormone-mediated effects of these insecticides on the risk of cancer among women. It can be concluded that, through experimental models, the initiation of cancer can be studied by analyzing the steps that transform normal breast cells to malignant ones through certain substances, such as pesticides and estrogen. Such substances cause genomic instability, and therefore tumor formation in the animal, and signs of carcinogenesis in vitro. Cancer initiation has been associated with an increase in genomic instability, indicated by the inactivation of tumor-suppressor genes and activation of oncogenes in the presence of malathion, parathion, and estrogen. In the present study, a comprehensive summary of the impact of OPs in human and rat breast cancer, specifically their effects on the cell cycle, signaling pathways linked to epidermal growth factor, drug metabolism, and genomic instability in an MCF-10F estrogen receptor-negative breast cell line is provided.

Achyrocline satureioides compounds, achyrobichalcone and 3-O-methylquercetin, induce mitochondrial dysfunction and apoptosis in human breast cancer cell lines.

Natural products are a valuable source of new molecules and are important for drug discovery. Many chemotherapeutics currently in clinical use were originated from natural sources and are effective cytotoxic agents. In this study, we investigated the cytotoxic and pro-apoptotic effects of achyrobichalcone (ACB) and 3-O-methylquercetin (3OMQ), two novel compounds isolated from the Achyrocline satureioides plant. Because extracts from this plant have been shown to have anticancer activity in vitro, we evaluated ACB and 3OMQ using a human breast cancer cell line, MDA-MB-231, and a nontumorigenic human breast epithelial cell line, MCF-12A. We found that ACB demonstrates cytotoxic effects on MDA-MB-231 cells, but not MCF-12A cells. 3OMQ also demonstrated cytotoxic effects on MDA-MB-231 cells, but with lower selectivity compared to treated MCF-12A cells. Cell death by both compounds was associated with caspase-9 and caspase-3/7 activation. Using high-resolution respirometry, we found that ACB and 3OMQ were able to cause acute mitochondrial dysfunction in MDA-MB-231-treated cells. These results suggest that apoptosis in MDA-MB-231 cells is induced through the activation of the mitochondrial-dependent pathway. Collectively, these findings suggest that ACB is a strong candidate for further anticancer in vivo tests.

Determining the Relative Gene Expression Level of Hypoxia Related Genes in Different Cancer Cell Lines.

This study aims to identify the changes in the expression of hypoxia-inducible genes in seven different cancer cell lines that vary in their oxygen levels in an attempt to identify hypoxia biomarkers that can be targeted in therapy. Profiling of hypoxia inducible-gene expression of these different cancer cell lines can be used as baseline data for further studies. Human cancer cell lines obtained from the American Type Culture Collection were used; MCF7 breast cancer cells, PANC-1 pancreatic cancer cells, PC-3 prostate cancer cells, SHSY5Y neuroblastoma brain cancer cells, A549 lung cancer cells, and HEPG2 hepatocellular carcinoma. In addition, we used the MCF10A non-tumorigenic human breast epithelial cell line as a normal cell line. The differences in gene expression were examined using real-time PCR array (PAHS- 032Z, Human Hypoxia Signaling Pathway PCR Array) and analyzed using the ΔΔCt method. Almost all hypoxia-inducible genes showed a PO2-dependent up- and down-regulated expression. Noticeable gene expression differences were identified. The most important changes occurred in the HIF1α and NF-KB signaling pathways targeted genes and in central carbon metabolism pathway genes such as HKs, PFKL, and solute transporters. This study identified possible hypoxia biomarkers genes such as NF-KB, HIF1α, HK, PFKL, and PIM1 that were expressed in all hypoxic cells. Pleiotropic pathways that play a role in inducing hypoxia directly, such as HIF1 α and NF-kB pathways, were upregulated. In addition, genes expressed only in the severe hypoxic liver and pancreatic cells indicate that severe and intermediate hypoxic cancer cells vary in their gene expression. Gene expression differences between cancer and normal cells showed the shift in gene expression profile to survive and proliferate under hypoxia.

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.

The halogenation of natural flavonoids, baicalein and chrysin, enhances their affinity to human protein kinase CK2.

A series of halogenated derivatives of natural flavonoids: baicalein and chrysin were designed and investigated as possible ligands for the catalytic subunit of tumor-associated human kinase CK2. Thermal shift assay method, in silico modeling, and high-performance liquid chromatography-derived hydrophobicity together with IC50 values determined in biochemical assay were used to explain the ligand affinity to the catalytic subunit of human protein kinase CK2. Obtained results revealed that substitution of baicalein and chrysin with halogen atom increases their binding affinity to hCK2α, and for 8-chlorochrysin the observed effect is even stronger than for the reference CK2 inhibitor-4,5,6,7-tetrabromo-1H-benzotriazole. The cytotoxic activities of the baicalein and chrysin derivatives in the in vitro model have been evaluated for MV4-11 (human biphenotypic B myelomonocytic leukemia), A549 (human lung adenocarcinoma), LoVo (human colon cancer), and MCF-7 (human breast cancer) as well as on the nontumorigenic human breast epithelial MCF-10A cell lines. Among the baicalein derivatives, the strongest cytotoxic effect was observed for 8-bromobaicalein, which exhibited the highest activity against breast cancer cell line MCF-7 (IC50 10 ± 3 μM). In the chrysin series, the strongest cytotoxic effect was observed for unsubstituted chrysin, which exhibited the highest activity against leukemic cell line MV4-11 (IC50 10 ± 4 μM).

Silver(I) metallodrugs of thiosemicarbazones and naproxen: biocompatibility, in vitro anti-proliferative activity and in silico interaction studies with EGFR, VEGFR2 and LOX receptors.

Four new heteroleptic silver(I) complexes with the general formula [Ag(L1-4)(nap)] (1-4), where L1-4= 2-(1-(4-substitutedphenyl)ethylidene)hydrazinecarbothioamide and nap=naproxen, have been synthesized and characterized. The geometric parameters determined from density functional theory and UV-Vis studies indicate distorted tetrahedral geometry around silver(I) ion. Fourier transform infrared (FT IR) spectra evidenced asymmetric bidentate coordination mode of carboxyl oxygen atoms of naproxen with silver(I) ion. The complexes are stable for 72h and biocompatibility was analysed towards normal human dermal fibroblast cells, which showed non-toxic nature up to 100ng/ml. In vitro anti-proliferative activity of the complexes by MTT assay was tested against three human cancerous cell lines and one non-tumorigenic human breast epithelial cell line (MCF-10a) in which the complex 4 exhibited enhanced activity. The morphological changes observed by acridine orange/ethidium bromide and Hoechst 33258 staining method reveal apoptosis-inducing ability of the complexes. The molecular docking studies suggest hydrogen bonding, hydrophobic and π-pair interactions with the active site of epidermal growth factor receptor, vascular endothelial growth factor receptor 2 and lipoxygenase receptors.

Proteomics of transforming growth factorβ1(TGFβ1) signaling in 184A1 human breast epithelial cells suggests the involvement of casein kinase2α inTGFβ1-dependent p53 phosphorylation at Ser392.

Transforming growth factorβ1 (TGFβ1) is a potent regulator of breast tumorigenesis. It inhibits proliferation of carcinoma cells, but the strength of its inhibitory action varies for cells from benigh, non-metastatic or metastatic tumors. The aim of this work was to generate a proteome profile of TGFβ1action on non-tumorigenic human breast epithelial cells 184A1, and validate predicted involvement of casein kinase 2α (CK2α), p53and structure-specific recognition protein-1 (SSRP1). Two-dimensional gel electrophoresis and mass spectrometry were used to identify TGF β1-regulated proteins in 184A1human breast immortalized non-tumorigenic cells. 184A1cells may serve as a model of benign breast neoplasia. These cells were obtained from normal mammary tissue, were immortalized but are not malignant, and were obtained from the American Type Culture Collection. The systemic analysis was performed by using the Cytoscape tool. Transfection of cells with CK2α construct and small interfering RNAs to CK2α and SSRP1were used to assess an impact of CK2α and SSRP1on phosphorylation of the p53and cell proliferation. Proliferation of 184A1cells was transiently inhibited by TGF β1. We identified 100and 47unique proteins which changed their expression and/or 35S-incorporation, respectively, upon treatment with TGF β1for 2h, 8h or 24h. Cell proliferation, death, migration, and metabolism were among the biological regulatory processes retrieved by the network analysis as affected by the identified proteins. The network analysis suggested that TGF β1may affect the phosphorylation of p53at Ser392by engaging CK2α. This was confirmed by the immunoblotting and cell proliferation assays. We report here the list of 147TGF β1-regulated proteins in immortalized non-tumorigenic human breast epithelial cells, and show involvement of CK2α in the regulation of p53Ser392phosphorylation.

Proteomics of transforming growth factor β1 (TGF β1) signaling in 184A1 human breast epithelial cells suggests the involvement of casein kinase 2α in TGF β1-dependent p53 phosphorylation at Ser392

Summary. Aim: Transforming growth factor β1 (TGF β1) is a potent regulator of breast tumorigenesis. It inhibits proliferation of carcinoma cells, but the strength of its inhibitory action varies for cells from benigh, non-metastatic or metastatic tumors. The aim of this work was to generate a proteome profile of TGF β1 action on non-tumorigenic human breast epithelial cells 184A1, and validate predicted involvement of casein kinase 2α (CK2α), p53 and structure-specific recognition protein-1 (SSRP1). Materials and Methods: Two-dimensional gel electrophoresis and mass spectrometry were used to identify TGF β1-regulated proteins in 184A1 human breast immortalized non-tumorigenic cells. 184A1 cells may serve as a model of benign breast neoplasia. These cells were obtained from normal mammary tissue, were immortalized but are not malignant, and were obtained from the American Type Culture Collection. The systemic analysis was performed by using the Cytoscape tool. Transfection of cells with CK2α construct and small interfering RNAs to CK2α and SSRP1 were used to assess an impact of CK2α and SSRP1 on phosphorylation of the p53 and cell proliferation. Results: Proliferation of 184A1 cells was transiently inhibited by TGF β1. We identified 100 and 47 unique proteins which changed their expression and/or 35S-incorporation, respectively, upon treatment with TGF β1 for 2 h, 8 h or 24 h. Cell proliferation, death, migration, and metabolism were among the biological regulatory processes retrieved by the network analysis as affected by the identified proteins. The network analysis suggested that TGF β1 may affect the phosphorylation of p53 at Ser392 by engaging CK2α. This was confirmed by the immunoblotting and cell proliferation assays. Conclusion: We report here the list of 147 TGF β1-regulated proteins in immortalized non-tumorigenic human breast epithelial cells, and show involvement of CK2α in the regulation of p53 Ser392 phosphorylation.

Altered profiles and metabolism of l- and d-amino acids in cultured human breast cancer cells vs. non-tumorigenic human breast epithelial cells

Herein we describe for the first time the endogenous levels of free l-and d-amino acids in cultured human breast cancer cells (MCF-7) and non-tumorigenic human breast epithelial cells (MCF-10A). d-Asp and d-Ser, which are co-agonists of the N-methyl-d-aspartate (NMDA) receptors, showed significantly elevated levels in MCF-7 cancer cells compared to MCF-10A cells. This may result from upregulated enzymatic racemases. Possible roles of these d-amino acids in promoting breast cancer proliferation by regulating NMDA receptors were indicated. d-Asn may also be able to serve as exchange currency, like specific l-amino acids, for the required uptake of essential amino acids and other low abundance nonessential amino acids which were elevated nearly 60 fold in cancer cells. The relative levels of specific l- and d-amino acids can be used as malignancy indicators (MIs) for the breast cancer cell line in this study. High MIs (>50) result from the increased demands of specific essential amino acids. Very low MIs (<1) result from the increased demands of specific d-amino acids (i.e., d-Ser, d-Asp) or the cellular release of amino acid exchange currency (i.e., l- and d-Asn) used in the upregulated amino acid antiporters to promote cancer cell proliferation.

Nitro-fatty acid inhibition of triple-negative breast cancer cell viability, migration, invasion, and tumor growth

Triple-negative breast cancer (TNBC) comprises ∼20% of all breast cancers and is the most aggressive mammary cancer subtype. Devoid of the estrogen and progesterone receptors, along with the receptor tyrosine kinase ERB2 (HER2), that define most mammary cancers, there are no targeted therapies for patients with TNBC. This, combined with a high metastatic rate and a lower 5-year survival rate than for other breast cancer phenotypes, means there is significant unmet need for new therapeutic strategies. Herein, the anti-neoplastic effects of the electrophilic fatty acid nitroalkene derivative, 10-nitro-octadec-9-enoic acid (nitro-oleic acid, NO2-OA), were investigated in multiple preclinical models of TNBC. NO2-OA reduced TNBC cell growth and viability in vitro, attenuated TNFα-induced TNBC cell migration and invasion, and inhibited the tumor growth of MDA-MB-231 TNBC cell xenografts in the mammary fat pads of female nude mice. The up-regulation of these aggressive tumor cell growth, migration, and invasion phenotypes is mediated in part by the constitutive activation of pro-inflammatory nuclear factor κB (NF-κB) signaling in TNBC. NO2-OA inhibited TNFα-induced NF-κB transcriptional activity in human TNBC cells and suppressed downstream NF-κB target gene expression, including the metastasis-related proteins intercellular adhesion molecule-1 and urokinase-type plasminogen activator. The mechanisms accounting for NF-κB signaling inhibition by NO2-OA in TNBC cells were multifaceted, as NO2-OA (a) inhibited the inhibitor of NF-κB subunit kinase β phosphorylation and downstream inhibitor of NF-κB degradation, (b) alkylated the NF-κB RelA protein to prevent DNA binding, and (c) promoted RelA polyubiquitination and proteasomal degradation. Comparisons with non-tumorigenic human breast epithelial MCF-10A and MCF7 cells revealed that NO2-OA more selectively inhibited TNBC function. This was attributed to more facile mechanisms for maintaining redox homeostasis in normal breast epithelium, including a more favorable thiol/disulfide balance, greater extents of multidrug resistance protein-1 (MRP1) expression, and greater MRP1-mediated efflux of NO2-OA-glutathione conjugates. These observations reveal that electrophilic fatty acid nitroalkenes react with more alkylation-sensitive targets in TNBC cells to inhibit growth and viability.

Pro-apoptotic effect of Δ2-TGZ in “claudin-1-low” triple-negative breast cancer cells: involvement of claudin-1

40% of triple-negative breast cancer (TNBC) do not express claudin-1, a major constituent of tight junction. Patients with these "claudin-1-low" tumors present a higher relapse incidence. A major challenge in oncology is the development of innovative therapies for such poor prognosis tumors. In this context, we study the anticancer effects of ∆2-TGZ, a compound derived from troglitazone (TGZ), on cell models of these tumors. In MDA-MB-231 and Hs578T "claudin-1-low" TNBC cells, Δ2-TGZ treatment induced claudin-1 protein expression and triggered apoptosis as measured by FACS analysis (annexin V/PI co-staining). Interestingly, in the non-tumorigenic human breast epithelial cell line MCF-10A, the basal level of claudin-1 was not modified following Δ2-TGZ treatment, which did not induce apoptosis. Furthermore, claudin-1-transfected MDA-MB-231 and Hs578T cells displayed a significant increase of cleaved PARP-1 and caspase 7, caspase 3/7 activities, and TUNEL staining. RNA interference was performed in order to inhibit Δ2-TGZ-induced claudin-1 expression in both the cells. In absence of claudin-1, a decrease of cleaved PARP-1 and caspase 7 and caspase 3/7 activities were observed in MDA-MB-231 but not in Hs578T cells. Claudin-1 overexpression and Δ2-TGZ treatment are associated to apoptosis in MDA-MB-231 and Hs578T "claudin-1-low" TNBC. Moreover, in MDA-MB-231 cells, claudin-1 is involved in the pro-apoptotic effect of Δ2-TGZ. Our results suggest that claudin-1 re-expression could be an interesting therapeutic strategy for "claudin-1-low" TNBC.

Abstract 1772: PIK3CA C2 domain deletions hyperactivate PI3K, generate oncogene dependence and are exquisitely sensitive to PI3Kα inhibitors

Abstract Background: A 63 year old postmenopausal woman with advanced ER+ breast cancer resistant to endocrine therapy exhibited an exceptional response to the PI3Kα inhibitor alpelisib (BYL719) and the aromatase inhibitor letrozole (Mayer et al. Clin Cancer Res 2016). Targeted capture next generation sequence (NGS) of DNA from a liver metastasis identified a P447_L455 deletion in the C2 domain of PIK3CA. About 80% of PIK3CA activating mutations are in ‘hot spots’ within the helical and kinase domains. C2 domain mutations make up ~10% of all PIK3CA mutations in breast cancer (TCGA, Foundation Medicine) and are frequently not reported by tumor and plasma cell-free DNA NGS panels. Deletions and mutations in this domain cluster in a region encompassing amino acids 446-460 of PIK3CA. We investigated herein the functional role of PIK3CA C2 domain mutations and their response to PI3K inhibitors. Methods: V5-tagged lentiviral vectors encoding wild type, delP447-L455, and delH450-P458 were stably transduced into MCF10A non-tumorigenic human breast epithelial cells. Cell viability and acini formation in 3D Matrigel were examined in media ± EGF or insulin or alpelisib. Markers of PI3K activation were examined by immunoblot and phosphoflow analysis. The Rosetta software suite was used to construct a structural model that would predict the change in stability of the p85/p110α complex. Physical association of p85α and p110α was determined by precipitation with V5 antibodies and immunoblot analysis. Results: MCF10A cells stably expressing V5-tagged PIK3CAdelP447-L455 and PIK3CAdelH450-P458 exhibited EGF- and insulin-independent growth and higher phosphorylation of AKT, ERK and S6 when compared to parental MCF10A cells. In 3D Matrigel, MCF10A cells with PIK3CA C2 domain deletions formed invasive acini with increased protrusions, spindling, and bridging between acini. All these changes were ablated upon the addition of 1 µM of alpelisib whereas parental MCF10A cells were unaffected by alpelisib. We hypothesized that delP447-L455 would reduce the binding affinity of p110α with the p85 regulatory subunit of PI3K. A structural model of PIK3CAdelP447-L455 in the context of the regulatory complex revealed specific favorable inter-residue contacts that would be lost as a result of the deletion, predicting a significant decrease in binding energy. Consistent with this structural analysis, coimmunoprecipitation of p85 with V5 antibodies showed reduced binding of the C2 domain deletion mutants with p85 compared to wild type p110α. Conclusions: These data suggest that C2 domain deletions in PIK3CA are activating mutations and generate oncogene dependence. As a result, tumors expressing these mutations are exquisitely sensitive to PI3Kα inhibitors. Thus, in addition to PIK3CA ‘hot spot’ mutations, C2 domain mutations should also be considered biomarkers of sensitivity to PI3K inhibitors. Citation Format: Sarah Croessmann, Jonathan Sheehan, Gregory Sliwoski, Nalin Leelatian, Jie He, Rebecca Nagy, Justin M. Balko, Ingrid A. Mayer, Richard B. Lanman, Vincent Miller, Lewis C. Cantley, Jonathan M. Irish, Jens Meiler, Carlos L. Arteaga. PIK3CA C2 domain deletions hyperactivate PI3K, generate oncogene dependence and are exquisitely sensitive to PI3Kα inhibitors [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 1772. doi:10.1158/1538-7445.AM2017-1772

A new l-amino acid oxidase from Bothrops jararacussu snake venom: Isolation, partial characterization, and assessment of pro-apoptotic and antiprotozoal activities

A new l-amino acid oxidase (LAAO) from Bothrops jararacussu venom (BjussuLAAO-II) was isolated by using a three-step chromatographic procedure based on molecular exclusion, hydrophobicity, and affinity. BjussuLAAO-II is an acidic enzyme with pI=3.9 and molecular mass=60.36kDa that represents 0.3% of the venom proteins and exhibits high enzymatic activity (4884.53U/mg/mim). We determined part of the primary sequence of BjussuLAAO-II by identifying 96 amino acids, from which 34 compose the N-terminal of the enzyme (ADDRNPLEECFRETDYEEFLEIARNGLSDTDNPK). Multiple alignment of the partial BjussuLAAO-II sequence with LAAOs deposited in the NCBI database revealed high similarity (95–97%) with other LAAOs isolated from Bothrops snake venoms. BjussuLAAO-II exerted a strong antiprotozoal effect against Leishmania amazonensis (IC50=4.56μg/mL) and Trypanosoma cruzi (IC50=4.85μg/mL). This toxin also induced cytotoxicity (IC50=1.80μg/mL) and apoptosis in MCF7 cells (a human breast adenocarcinoma cell line) by activating the intrinsic and extrinsic apoptosis pathways, but were not cytotoxic towards MCF10A cells (a non-tumorigenic human breast epithelial cell line). The results reported herein add important knowledge to the field of Toxinology, especially for the development of new therapeutic agents.

Abstract P6-01-15: Novel regulation of breast cancer cell aggressiveness by cancer testis antigen

Abstract We recently demonstrated that a vesicular endocytosis associated protein SH3GL2, attenuates spontaneous metastases of breast cancer cells by inducing a mesenchymal to epithelial differentiation and the onset of the intrinsic apoptotic pathway. The present study aims to understand the molecular mechanism behind the SH3Gl2 mediated reduction of spontaneous metastasis of the breast cancer cells. We employed a cDNA microarray analysis of the SH3GL2-overexpressing breast cancer cells exhibiting reduced pulmonary metastasis and identified a 12.1 fold downregulation of SPANXB1, a cancer-testis antigen that regulates sperm motility. A limited number of studies reported an association between increased SPANXB1 expression and progression of melanoma and hepatocellular carcinoma. Augmented SPANXB1 mRNA and protein expression was evident in primary breast tumors and its upregulation was associated with pulmonary metastasis of breast cancer cells. However, the expression pattern of SPANXB1 and its role in BCa development and progression is unknown. By immunohistochemical analysis, we detected high expression (p=0.002) of SPANXB1 in 78% (18/23) of the primary breast cancer tissues and corresponding lymph node metastases compared to the matched normal breast tissues. A couple of non-tumorigenic human breast epithelial cell lines were stably transformed with SPANXB1 to understand its effect on cellular growth and progression. The SPANXB1-transformed cells exhibited increased invasion (p=0.0001) and epithelial to mesenchymal transition accompanied by an augmented expression ratio of Vimentin/E-Cadherin, molecules regulating differentiation and metastasis. The SPANXB1-transformed cells also exhibited a markedly reduced expression of SH3GL2, implicating a SPANXB1:SH3GL2 crosstalk accompanied by an enhanced production of lactate (p=0.004). Our investigation identifies new breast cancer promoting role of a cancer testis antigen, which bears potential for biomarker and targeted therapeutic development. Citation Format: Kannan A, Wells RM, Ikebe M, Dasgupta S. Novel regulation of breast cancer cell aggressiveness by cancer testis antigen [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P6-01-15.