Non-cancerous Mammary Epithelial Cells Research Articles

Extracellular vesicles as a promising source of lipid biomarkers for breast cancer detection in blood plasma.

Extracellular vesicles (EVs), including exosomes and microvesicles, mediate intercellular communication in cancer, from development to metastasis. EV-based liquid biopsy is a promising strategy for cancer diagnosis as EVs can be found in cancer patients' body fluids. In this study, the lipid composition of breast cancer-derived EVs was studied as well as the potential of blood plasma EVs for the identification of lipid biomarkers for breast cancer detection. Initially, an untargeted lipidomic analysis was carried out for a panel of cancerous and non-cancerous mammary epithelial cells and their secreted EVs. We found that breast cancer-derived EVs are enriched in sphingolipids and glycerophospholipids compared to their parental cells. The initial in vitro study showed that EVs and their parental cells can be correctly classified (100% accuracy) between cancerous and non-cancerous, as well as into their respective breast cancer subtypes, based on their lipid composition. Subsequently, an untargeted lipidomic analysis was carried out for blood plasma EVs from women diagnosed with breast cancer (primary or progressive metastatic breast cancer) as well as healthy women. Correspondingly, when blood plasma EVs were analysed, breast cancer patients and healthy women were correctly classified with an overall accuracy of 93.1%, based on the EVs' lipid composition. Similarly, the analysis of patients with primary breast cancer and healthy women showed an overall accuracy of 95% for their correct classification. Furthermore, primary and metastatic breast cancers were correctly classified with an overall accuracy of 89.5%. This reveals that the blood plasma EVs' lipids may be a promising source of biomarkers for detection of breast cancer. Additionally, this study demonstrates the usefulness of untargeted lipidomics in the study of EV lipid composition and EV-associated biomarker discovery studies. This is a proof-of-concept study and a starting point for further analysis on the identification of EV-based biomarkers for breast cancer.

Phytochemicals from Pterocarpus angolensis DC and Their Cytotoxic Activities against Breast Cancer Cells.

Pterocarpus anglonesis DC is an indigenous medicinal plant belonging to the Pterocarpus genus of the Fabaceae family. It is used to treat stomach problems, headaches, mouth ulcers, malaria, blackwater fever, gonorrhea, ringworm, diarrhea, heavy menstruation, and breast milk stimulation. Column chromatography of the stem bark extracts resulted in the isolation of eight compounds, which included friedelan-3-one (1), 3α-hydroxyfriedel-2-one (2), 3-hydroxyfriedel-3-en-2-one (3), lup-20(29)-en-3-ol (4), Stigmasta-5-22-dien-3-ol (5), 4-O-methylangolensis (6), (3β)-3-acetoxyolean-12-en-28-oic acid (7), and tetradecyl (E)-ferulate (8). The structures were established based on NMR, IR, and MS spectroscopic analyses. Triple-negative breast cancer (HCC70), hormone receptor-positive breast cancer (MCF-7), and non-cancerous mammary epithelial cell lines (MCF-12A) were used to test the compounds' cytotoxicity. Overall, the compounds showed either no toxicity or very low toxicity to all three cell lines tested, except for the moderate toxicity displayed by lupeol (4) towards the non-cancerous MCF-12A cells, with an IC50 value of 36.60 μM. Compound (3β)-3-acetoxyolean-12-en-28-oic acid (7) was more toxic towards hormone-responsive (MCF-7) breast cancer cells than either triple-negative breast cancer (HCC70) or non-cancerous breast epithelial (MCF-12A) cells (IC50 values of 83.06 vs. 146.80 and 143.00 μM, respectively).

Cancer Cell Membrane Wrapped Nanoparticles for the Delivery of a Bcl-2 Inhibitor to Triple-Negative Breast Cancer.

Overexpression of the antiapoptotic protein B-cell lymphoma 2 (Bcl-2) is correlated with poor survival outcomes in triple-negative breast cancer (TNBC), making Bcl-2 inhibition a promising strategy to treat this aggressive disease. Unfortunately, Bcl-2 inhibitors developed to date have limited clinical success against solid tumors, owing to poor bioavailability, insufficient tumor delivery, and off-target toxicity. To circumvent these problems, we loaded the Bcl-2 inhibitor ABT-737 in poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) that were wrapped with phospholipid membranes derived from 4T1 murine mammary cancer cells, which mimic the growth and metastasis of human TNBC. We show that the biomimetic cancer cell membrane coating enabled the NPs to preferentially target 4T1 TNBC cells over noncancerous mammary epithelial cells in vitro and significantly increased NP accumulation in orthotopic 4T1 tumors in mice after intravenous injection by over 2-fold compared to poly(ethylene glycol)-poly(lactide-co-glycolic) (PEG-PLGA) copolymer NPs. Congruently, the ABT-737 loaded, cancer cell membrane-wrapped PLGA NPs (ABT CCNPs) induced higher levels of apoptosis in TNBC cells in vitro than ABT-737 delivered freely or in PEG-PLGA NPs. When tested in a syngeneic spontaneous metastasis model, the ABT CCNPs significantly increased apoptosis (evidenced by elevated active caspase-3 and decreased Bcl-2 staining) and decreased proliferation (denoted by reduced Ki67 staining) throughout tumors compared with saline or ABT-loaded PEG-PLGA NP controls. Moreover, the ABT CCNPs did not alter animal weight or blood composition, suggesting that the specificity afforded by the TNBC cell membrane coating mitigated the off-target adverse effects typically associated with ABT-737. Despite these promising results, the low dose of ABT CCNPs administered only modestly reduced primary tumor growth and metastatic nodule formation in the lungs relative to controls. We posit that increasing the dose of ABT CCNPs, altering the treatment schedule, or encapsulating a more potent Bcl-2 inhibitor may yield more robust effects on tumor growth and metastasis. With further development, drug-loaded biomimetic NPs may safely treat solid tumors such as TNBC that are characterized by Bcl-2 overexpression.

CXCR4-Targeted Necrosis-Inducing Peptidomimetic for Treating Breast Cancer.

Triple-negative breast cancer is an aggressive subtype with a high recurrence rate, potential for metastasis, and a poor prognosis. The chemokine receptor, CXCR4, is a promising molecular target in breast cancer therapy. Here, we have developed a CXCR4-targeted antitumor peptidomimetic (named CTCE-KLAK), which is a fusion of the CXCR4 receptor antagonist CTCE-9908 and the D-form of proapoptotic peptide (KLAKLAK)2, for the treatment of breast cancer. First, we investigated the in vitro antitumor activity of CTCE-KLAK against various breast cancer cells and noncancerous mammary epithelial cells. CTCE-KLAK showed cell-selective cytotoxicity and induced rapid necrotic cell death in breast cancer cells but not in normal cells. In contrast, unconjugated peptides such as the carboxylate analogues of CTCE-9908 and D(KLAKLAK)2 were not cytotoxic to these cells. The tumor selectivity of CTCE-KLAK for cytotoxic activity depends on its internalization into tumor cells. There was no cleavage of caspase-3, caspase-7, or PARP1 in CTCE-KLAK-treated cells. In addition, cell death by CTCE-KLAK was not prevented by z-VAD-fmk, a pan-caspase inhibitor that inhibits cisplatin-induced cell death. These data indicate that the CTCE-KLAK conjugate is a cell-selective inducer of necrosis. Furthermore, we evaluated the in vivo antitumor activity of CTCE-KLAK in the 4T1 mouse metastatic breast cancer model. Intravenous administration of CTCE-KLAK significantly inhibited tumor growth and lung metastasis. Together, these findings suggest that the necrosis-inducing peptidomimetic CTCE-KLAK is a promising CXCR4-targeted agent for treating triple-negative breast cancer.

Epigenetic Dysregulation of KCNK9 Imprinting and Triple-Negative Breast Cancer.

Simple SummaryGenomic imprinting is an inherited form of parent-of-origin specific epigenetic gene regulation that is dysregulated by poor prenatal nutrition and environmental toxins. Here, we showed that KCNK9 is imprinted in breast tissue and identified the differentially methylated region (DMR) controlling its imprint status. Hypomethylation at the DMR, coupled with biallelic expression of KCNK9, occurred in 63% of triple-negative breast cancers (TNBC). The association between hypomethylation and TNBC status was highly significant in African-Americans (p = 0.006), but not in Caucasians (p = 0.70). The high frequency of KCNK9 DMR hypomethylation in TNBC and non-cancerous breast tissue from high-risk women provides evidence that hypomethylation of the KNCK9 DMR/TASK3 overexpression may provide a new target for prevention of TNBC.Genomic imprinting is an inherited form of parent-of-origin specific epigenetic gene regulation that is dysregulated by poor prenatal nutrition and environmental toxins. KCNK9 encodes for TASK3, a pH-regulated potassium channel membrane protein that is overexpressed in 40% of breast cancer. However, KCNK9 gene amplification accounts for increased expression in <10% of these breast cancers. Here, we showed that KCNK9 is imprinted in breast tissue and identified a differentially methylated region (DMR) controlling its imprint status. Hypomethylation at the DMR, coupled with biallelic expression of KCNK9, occurred in 63% of triple-negative breast cancers (TNBC). The association between hypomethylation and TNBC status was highly significant in African-Americans (p = 0.006), but not in Caucasians (p = 0.70). KCNK9 hypomethylation was also found in non-cancerous tissue from 77% of women at high-risk of developing breast cancer. Functional studies demonstrated that the KCNK9 gene product, TASK3, regulates mitochondrial membrane potential and apoptosis-sensitivity. In TNBC cells and non-cancerous mammary epithelial cells from high-risk women, hypomethylation of the KCNK9 DMR predicts for increased TASK3 expression and mitochondrial membrane potential (p < 0.001). This is the first identification of the KCNK9 DMR in mammary epithelial cells and demonstration that its hypomethylation in breast cancer is associated with increases in both mitochondrial membrane potential and apoptosis resistance. The high frequency of hypomethylation of the KCNK9 DMR in TNBC and non-cancerous breast tissue from high-risk women provides evidence that hypomethylation of the KNCK9 DMR/TASK3 overexpression may serve as a marker of risk and a target for prevention of TNBC, particularly in African American women.

Geometry encoded functional programming of tumor homing peptides for targeted drug delivery

Poly-peptide molecules have shown promising applications in drug delivery and tumor targeting. A series of tumor homing peptides were designed by exhaustively sampling low energy geometrical basins of amino acids at specific sites of a peptide molecule to induce a conformational lock. This peptide library was pruned to a limited set of eight molecules, employing electrostatic interactions, docking, and molecular dynamics simulations. These designed and optimized peptides were synthesized and tested on various cell lines, including breast cancer (MDA-MB-231), cervical cancer (HeLa), osteosarcoma (U2-OS), and non-cancerous mammary epithelial cells (MCF-10A) using confocal microscopy and flow cytometry. Peptides show differential uptake in cancerous MDA-MB-231, HeLa, U2-OS, and non-cancerous MCF-10A cells. Confocal imaging verified their ability to penetrate even in 3D tumorospheres of MDA-MB-231 cells. Further, experiments of mitochondrial membrane potential depolarization and Caspase-3 activation confirmed that their cytotoxic effects are by apoptosis. Homing ability of the designed peptides in in vivo system and fluorescence imaging with clinical samples of human origin have further confirmed that the in vitro studies are qualitatively identical and quantitatively comparable in their ability to selectively recognize tumor cells. Overall, we present a roadmap for the functional programming of peptide-based homing and penetrating molecules that can perform selective tumor targeting.

Abstract 5918: The role of the cell-adhesion molecule Metadherin in inflammatory breast cancer progression

Abstract Inflammatory breast cancer (IBC) is the most lethal form of breast cancer where patients display a lower 5-year survival rate. The lethality of IBC originates from its nature of invading the vascular and lymphatic systems, absence of a tumor mass, and generation of tumor emboli. Currently, the accurate and early diagnosis and the development of effective targeted therapies remains a challenge. Thus, in an attempt to discover potential biomarkers, we characterized the IBC cell surface proteome. Plasma membrane proteins were identified via SILAC and MS quantitative proteomics using the IBC SUM-149 vs. MCF-10A a non-cancerous mammary epithelial cell line. SILAC results showed overexpression of Metadherin (MTDH) in IBC cells (&amp;gt;2.0 FCh) and was validated in IBC tissues and emboli. MTDH is a cell adhesion molecule overexpressed in many cancer types, including breast cancer. MTDH promotes cancer progression by modulating various signaling pathways including NF-kB, which is related to cell survival, proliferation, invasion and metastasis. Thus, the aim of this study is to assess the functional role of MTDH in IBC progression. To elucidate the mechanism of action of MTDH in colony formation and tumor spheroids we silenced MTDH (CRISPR or shRNA) SUM-149 and SUM-190 IBC cells. We assessed the expression of MTDH by immunoblotting and immunofluorescence where we observed reduced MDTH levels. The number of colonies formed in SUM-149 MTDH silenced cells was ~40% lower when compared to non-silenced cells. Tumor spheroids of SUM-149 and SUM-190 MTDH silenced cells were smaller than non-silenced cells. Additionally, MTDH silencing results in a decrease in the phosphorylation of the p65 subunit of NF-kB while no changes are observed in total protein abundance in SUM-149 cells. We can conclude from our preliminary results that MTDH serves as a potential IBC target for IBC progression. This work was supported by NIH NIGMS #GM111171 (MMM), SGRP 2017-00143 (MMM and GOS), NIMHD #MD007583 (MMM), GM103475 (UPR MMM), Title-V-PPOHA #P031M105050, Title-V-Cooperative #P031S130068 from the U.S. Dept. of Education and Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health #P20GM103475 (GOS). Citation Format: Gabriela Ortiz-Soto, Ivette J. Suárez-Arroyo, Michelle M. Martínez-Montemayor. The role of the cell-adhesion molecule Metadherin in inflammatory breast cancer progression [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 5918.

Cytotoxicity of triterpenoids from Clerodendrum glabrum against triple negative breast cancer cells in vitro

Clerodendrum glabrum is an indigenous medicinal plant that is used to treat cough, cold, sore throat and chest complaints. The stem bark of Clerodendrum glabrum afforded four tritepenoids namely, 3β-olean-12-en-3-yl palmitate (β-amyrin palmitate), (1), 3β‑hydroxy‑5-glutinene (glutinol), (2), 3β-lup-20(29)-en-3-palmitate (Lupeol-3-palmitate), (3), 3β-lup-20(29)-en-3-ol (lupeol) (4) and one common phytosterol (stigmasterol) (5). The structures were established on the basis of their spectroscopic analysis. The compounds were screened for cytotoxicity against the HCC70 triple negative breast cancer (TNBC), MCF-7 hormone receptor positive breast cancer and MCF-12A non-cancerous mammary epithelial cell lines. Interestingly, none of the compounds were toxic towards hormone receptor positive breast cancer cells, while displaying varying toxicity against the TNBC and non-cancerous breast epithelial cells. In particular, lupeol-3-palmitate (47.6 ± 1.50 μM) and glutinol (26.9 ± 1.30 μM) displayed the greatest inhibitory activity against the HCC70 cell line, with the former being selectively toxic to HCC70 and not MCF-12A non-cancerous cells.

An Inhibitor of Fatty Acid Synthase Thioesterase Domain with Improved Cytotoxicity against Breast Cancer Cells and Stability in Plasma.

It is well recognized that many cancers are addicted to a constant supply of fatty acids (FAs) and exhibit brisk de novo FA synthesis. Upregulation of a key lipogenic enzyme, fatty acid synthase (FASN), is a near-universal feature of human cancers and their precursor lesions, and has been associated with chemoresistance, tumor metastasis, and diminished patient survival. FASN inhibition has been shown to be effective in killing cancer cells, but progress in the field has been hindered by off-target effects and poor pharmaceutical properties of candidate compounds. Our initial hit (compound 1) was identified from a high-throughput screening effort by the Sanford-Burnham Center for Chemical Genomics using purified FASN thioesterase (FASN-TE) domain. Despite being a potent inhibitor of purified FASN-TE, compound 1 proved highly unstable in mouse plasma and only weakly cytotoxic to breast cancer (BC) cells in vitro. An iterative process of synthesis, cytotoxicity testing, and plasma stability assessment was used to identify a new lead (compound 41). This lead is more cytotoxic against multiple BC cell lines than tetrahydro-4-methylene-2S-octyl-5-oxo-3R-furancarboxylic acid (the literature standard for inhibiting FASN), is stable in mouse plasma, and shows negligible cytotoxic effects against nontumorigenic mammary epithelial cells. Compound 41 also has drug-like physical properties based on Lipinski's rules and is, therefore, a valuable new lead for targeting fatty acid synthesis to exploit the requirement of tumor cells for fatty acids. SIGNIFICANCE STATEMENT: An iterative process of synthesis and biological testing was used to identify a novel thioesterase domain FASN inhibitor that has drug-like properties, is more cytotoxic to breast cancer cells than the widely used tetrahydro-4-methylene-2S-octyl-5-oxo-3R-furancarboxylic acid, and has negligible effects on the growth and proliferation of noncancerous mammary epithelial cells. Our studies have confirmed the value of using potent and selective FASN inhibitors in the treatment of BC cells and have shown that the availability of exogenous lipoproteins may impact both cancer cell FA metabolism and survival.

SUN-010 BRCA1 Mutation Influences Progesterone Response in Human Benign Mammospheres

Women, who carry a germline mutation in the breast cancer-associated gene 1 (BRCA1) have an increased risk (60-85%) of developing breast cancer during their lifetime. While BRCA1 mutated patients usually develop triple-negative, basal like, aggressive breast tumors, hormone signaling is important in the genesis of BRCA1 mutant breast cancers. Estrogen and progesterone promote carcinogenesis in the breast. Progesterone promotes proliferation of both benign epithelial cells and cancer cells and can expand the mammary stem cell pool. It is unclear how PR functions in the background of BRCA1 mutation in normal non-cancerous mammary epithelial cells before breast cancer develops. In order to determine whether BRCA1 mutation affects hormone response in benign mammary cells, human mammospheres from patients with wild type (WT; n=6) or BRCA1 mutation (n=6) were treated in vitro with a stepwise menstrual cycle hormone regimen of estrogen (E2) and progesterone (P4) over the course of 28 days. During the last 14 days of treatment, the SPRM, telapristal acetate (TPA) was added to the mammospheres. The mammospheres grown from benign breast tissues retained the expression of hormone receptors (ER and PR) even after the 28 day hormone treatment in vitro. Human mammary epithelial cells expressed aldehyde dehydrogenese-1 ALDH-1 with increasing levels observed in the BRCA1 mutated mammospheres compared to the WT mammospheres. RNA sequencing (RNA-seq) analysis revealed distinct differentially expressed genes between the WT and BRCA1 groups. Specifically, genes associated with extracellular matrix (ECM) organization were differentially regulated by TPA in the BRCA1 mutation group but not in the WT group. In fact, these ECM genes were differentially expressed in response to E2+P4 in WT versus BRCA1 mammospheres. In addition, cell cycle genes were responsive to TPA but in the WT group only. Taken together, these data show that BRCA1 mutation influences the hormone response and in particular to TPA compared to WT cells. Our physiologically relevant model systems have provided important insights into the role of progesterone in BRCA1 mutated breast cells. Further analysis of the molecular mechanism of BRCA1 and PR crosstalk is warranted using this model system.

Abstract 4185: The novel Rac inhibitor HV-107 as a potential therapeutic for metastatic breast cancer

Abstract Breast cancer is the first cause of death in women globally. Metastatic breast cancer, which occurs when cancer cells migrate from the primary tumor and establish in distant organs, is estimated to affect more than a quarter of a million of women in the US. Regrettably, metastatic breast cancer still lacks effective treatment and remains the principal cause of breast cancer mortality. Therefore, it is imperative to develop new and more effective strategies to inhibit metastasis. A promising target for anti-metastatic cancer therapy is the Rho GTPase Rac because it plays a key role in the regulation of cellular adhesion, migration, proliferation and survival; processes which promote tumorigenesis and metastasis. Recently, our group developed Ehop-016, a small molecule that inhibits Rac in metastatic breast cancer cells with an IC50 of 1µM and reduces the activity of its downstream effector p21-activated kinase (PAK). Ehop-016 also inhibits tumor growth, angiogenesis, and metastasis in a mouse model of metastatic breast cancer. However, its relative bioavailability is moderate and should be further improved. Therefore, in an effort to find a compound with greater inhibitory capacity and bioavailability, we tested several Ehop-016 derivatives for their activity against Rac and their toxicity towards metastatic breast cancer cells. Using Rac pulldown assays we show that HV-107 at 250nM inhibits Rac activation by 55% in MDA-MB-231 and MDA-MB-435 metastatic breast cancer cells. The effects of HV-107 on cell viability were tested by MTT assays and our results indicate HV-107 at ≥500nM significantly inhibits metastatic breast cancer cell viability, while showing minimal toxicity towards non-cancerous mammary epithelial cells. We also measured caspase 3 activity, in order to evaluate apoptosis as a potential mechanism of HV-107-induced cell death. Our results show HV-107 at 1000nM causes a significant 1.5 fold increase in caspase 3 activity in MDA-MB-231 cells, indicating HV-107 affects cell viability by inducing apoptosis. Finally, we tested HV-107 in a mouse model of metastatic breast cancer and found a 25% reduction in tumor growth in mice treated with 5mg/kg BW HV-107. Taken together, our results suggest HV-107 is approximately four times more efficient than the parent compound Ehop-016 and has potential as anti-breast cancer metastasis therapeutic. This study was supported by awards from the Susan Komen for the Cure, NIH/NIMHHD U54MD008149, and the Puerto Rico Science and Technology Trust to SD; NIH/NCRR R25GM061838 to UPR MSC; NIH/NIMHHD RCMI 8G12MD007583RCMI, Title V PPOHA 031M10505 and Title V Cooperative P031S130068 from U.S. Department of Education to UCC; and PRINBRE (NIH/NIGMS P20GM103475-13) Sub-Award to LCP. Citation Format: Grace E. Velez Crespo, Eliud Hernández, Cornelis Vlaar, Suranganie Dharmawardhane, Linette Castillo-Pichardo. The novel Rac inhibitor HV-107 as a potential therapeutic for metastatic breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4185.

Comparison of the seleno-transcriptome expression between human non-cancerous mammary epithelial cells and two human breast cancer cell lines.

Breast cancer is the second most common cause of mortality in women; therefore, the identification of novel putative markers is required to improve its diagnosis and prognosis. Selenium is known to protect mammary epithelial cells from oxidative DNA damage, and to inhibit the initiation phase of carcinogenesis by stimulating DNA repair and apoptosis regulation. Consequently, the present study has focused attention on the selenoprotein family and their involvement in breast cancer. The present study performed a global analysis of the seleno-transcriptome expression in human breast cancer MCF-7 and MDA-MB231 cell lines compared with healthy breast MCF-10A cells using reverse transcription-quantitative polymerase chain reaction. The present data revealed the presence of differently expressed genes in MCF-7 and MDA-MB231 cells compared with MCF-10A cells: Four downregulated [glutathione peroxidase (GPX)1, GPX4, GPX5 and GPX7] and three upregulated (deiodinase iodothyronine, type II, GPX2 and GPX3) genes. Additionally, interactomic investigation were performed by the present study to evaluate the association between the downregulated and upregulated genes, and to identify putative HUB nodes, which represent the centers of association between the genes that are capable of direct control over the gene networks. Network analysis revealed that all differentially regulated genes, with the exception of selenoprotein T, are implicated in the same network that presents three HUB nodes interconnected to the selenoprotein mRNAs, including TP53, estrogen receptor 1 and catenin-β1 (CTNNB1). Overall, these data demonstrated for the first time, a profile of seleno-mRNAs specific for human breast cells, indicating that these genes alter their expression on the basis of the ER-positivity or negativity of breast cancer cells.

Activated-PAK4 predicts worse prognosis in breast cancer and promotes tumorigenesis through activation of PI3K/AKT signaling.

The p21-activated kinase 4 (PAK4) is sufficient to transform noncancerous mammary epithelial cells and to form tumors in the mammary glands of mice. The accumulated information suggests that PAK4 might be an oncogenic protein in breast cancer. In this study, we sought to identify the role for PAK4 in breast cancer progression. Immunohistochemical study revealed that high PAK4 expression is associated with larger tumor size, lymph node metastasis, and advanced stage cancer in 93 invasive breast carcinoma patients. Moreover, high PAK4 expression was significantly associated with poor overall and disease-free survival. PAK4 remained an independent adverse prognosticator after univariate and multivariate analysis. Ectopic expression of wild-type PAK4 in MDA-MB-231 cells activated PI3K/AKT signaling and resulted in the enhancement of the cell proliferation, migration, and invasion, whereas PAK4-induced effects were blocked by the PAK4 kinase inhibitor PF- 3758309, PAK4 siRNAs or the PI3K inhibitor LY294002. Furthermore, a kinase-active PAK4 (S474E) strongly induced PI3K/AKT activation, and promoted proliferation, migration and invasion in breast cancer cells. A kinase-inactive PAK4 KD (K350A/K351A) did partially upregulate PI3K/AKT, and promoted invasive phenotype. Taken together, these findings suggest that PAK4-activated PI3K/AKT signaling is both kinase-dependent and -independent, which contributes to breast cancer progression. Thus, our results imply that dual inhibition of PAK4 and PI3K/AKT signaling might be a potential therapeutic approach for breast cancer therapy.

Cross-kingdom inhibition of breast cancer growth by plant miR159.

MicroRNAs (miRNAs) are critical regulators of gene expression, and exert extensive impacts on development, physiology, and disease of eukaryotes. A high degree of parallelism is found in the molecular basis of miRNA biogenesis and action in plants and animals. Recent studies interestingly suggest a potential cross-kingdom action of plant-derived miRNAs, through dietary intake, in regulating mammalian gene expression. Although the source and scope of plant miRNAs detected in mammalian specimens remain controversial, these initial studies inspired us to determine whether plant miRNAs can be detected in Western human sera and whether these plant miRNAs are able to influence gene expression and cellular processes related to human diseases such as cancer. Here we found that Western donor sera contained the plant miRNA miR159, whose abundance in the serum was inversely correlated with breast cancer incidence and progression in patients. In human sera, miR159 was predominantly detected in the extracellular vesicles, and was resistant to sodium periodate oxidation suggesting the plant-originated 2'-O-methylation on the 3' terminal ribose. In breast cancer cells but not non-cancerous mammary epithelial cells, a synthetic mimic of miR159 was capable of inhibiting proliferation by targeting TCF7 that encodes a Wnt signaling transcription factor, leading to a decrease in MYC protein levels. Oral administration of miR159 mimic significantly suppressed the growth of xenograft breast tumors in mice. These results demonstrate for the first time that a plant miRNA can inhibit cancer growth in mammals.

Cellular uptake, genotoxicity and cytotoxicity of cobalt ferrite magnetic nanoparticles in human breast cells.

Magnetic nanoparticles (MNPs) have been increasingly used for many years as MRI agents and for gene delivery and hyperthermia therapy, although there have been conflicting results on their safety. In this study, cobalt ferrite magnetic nanoparticles (CoFe-MNPs) were prepared by the co-precipitation method and their surfaces were modified with silica by the sol-gel method. The particle and hydrodynamic sizes, morphology and crystal structure of the bare and silica-coated CoFe-MNPs were evaluated by transmission electron microscopy (TEM), dynamic light scattering (DLS), X-ray diffraction spectroscopy (XRD) and Fourier transform infrared spectroscopy (FTIR). The size of the bare CoFe-MNPs was in the range 8-20 nm and they were homogeneously coated with 3-4 nm silica shells. The bare and silica-coated CoFe-MNPs were agglomerated at physiological pH. However, the sizes of the agglomerates were below 200 nm both in water and complete medium. The cytotoxic and genotoxic potentials of the bare and silica-coated CoFe-MNPs were evaluated in a metastatic breast cancer cell line, MDA-MB-231, as well as a noncancerous mammary epithelial cell line, MCF-10A, by using XTT cytotoxicity, single-cell gel electrophoresis (comet), and cytokinesis-blocked (CB) micronucleus (CBMN) assays. Characterization studies with TEM, inductively coupled plasma optical emission spectroscopy (ICP-OES) and Prussian blue staining indicated that the CoFe-MNPs were internalized into the cells by energy-dependent endocytosis. The highest amount of uptake was observed in the cancer cells and the uptake of the silica-coated CoFe-MNPs was higher than that of the bare ones in both cell lines. The bare CoFe-MNPs showed higher levels of both cytotoxicity and genotoxicity than the silica-coated CoFe-MNPs. Moreover, the cancer cells seemed to be more susceptible to the CoFe-MNPs' toxicity compared to the noncancerous cells. There was a concentration and time-dependent increase in DNA damage and the micronucleus (MN) frequency, which was statistically significant starting with the lowest concentration of bare CoFe-MNPs (p < 0.05), while no significance was observed below the concentration of 250 μg mL-1 for the silica-coated MNPs. Also, the extent of both DNA damage and MN frequency was much higher in the cancer cells compared to the noncancerous cells. According to our results, the silica coating ameliorated both the cytotoxicity and genotoxicity as well the internalization of the CoFe-MNPs.

Normal mammary epithelial cells promote carcinoma basement membrane invasion by inducing microtubule-rich protrusions.

Recent work suggests that the dissemination of tumor cells may occur in parallel with, and even preceed, tumor growth. The mechanism for this early invasion is largely unknown. Here, we find that mammary epithelial cells (MECs) induce neighboring breast carcinoma cells (BCCs) to cross the basement membrane by secreting soluble laminin. Laminin continuously produced by MECs induce long membrane cellular protrusions in BCCs that promote their contractility and invasion into the surrounding matrix. These protrusions depend on microtubule bundles assembled de novo through laminin-integrin β1 signaling. These results describe how non-cancerous MECs can actively participate in the invasive process of BCCs.

Abstract 1717: Selective induction of breast adenocarcinoma cell death via inhibition of the transient receptor potential melastatin-2 (TRPM2) cation channel

Abstract Transient receptor potential melastatin-2 (TRPM2) is a plasma membrane cation channel with important physiologic roles in noncancerous cells. However, in MCF-7 and MDA-MB-231 human breast adenocarcinoma cells, we previously demonstrated that TRPM2 was present in the nucleus, and pharmacologic inhibition or RNAi silencing of TRPM2 led to decreased proliferation. In this study, we evaluated the ability of TRPM2 inhibition to produce cell death in normal breast epithelial cells and breast adenocarcinoma cells. Further, we analyzed the cell death pathways induced. Treatment with the DNA alkylating agent, N-methyl-N’-nitro-N-nitrosoguanidine (MNNG), or doxorubicin (Dox) led to increased levels of cell death in MDA-MB-231 breast adenocarcinoma cells pretreated with TRPM2 inhibitors, but not in noncancerous mammary epithelial cells after pretreatment. Similar increases in cell death were observed after RNAi silencing of TRPM2 in both MDA-MB-231 and MCF-7 breast adenocarcinoma cells after Dox or tamoxifen treatment, respectively. Investigation of apoptosis after TRPM2 inhibition demonstrated decreased levels of caspase activity and no significant effect of Q-VD-OPh, a pan-caspase inhibitor, on cell death induced by TRPM2 inhibition. Further, pretreatment with 3-methyl adenine, an inhibitor of autophagy, also produced no significant effect on cell death induced by TRPM2 inhibition. These studies indicate that apoptosis or autophagy are not the primary cell death pathways induced by TRPM2 inhibition in breast cancer cells. Also, RNAi silencing of poly(ADP-ribose) glycohydrolase (PARG) and apoptosis-inducing factor (AIF), two proteins involved in initiating or facilitating alternative pathways of cell death, produced minimal changes in cell death induced by TRPM2 inhibition. Taken together, this study demonstrated that TRPM2 inhibition selectively increases cell death in breast adenocarcinoma cells. Further, the data suggests that apoptosis, autophagy, and PARG/AIF-mediated cell death are not the primarily cell death pathways involved in the cytotoxicity caused by TRPM2 inhibition. The results therefore suggest that TRPM2 is a potential target in breast cancer, where its inhibition may cause the selective eradication of breast adenocarcinoma cells. Citation Format: David W. Koh, Daniel P. Powell, Steven D. Blake, Joy L. Hoffman, Xiaoxing Feng. Selective induction of breast adenocarcinoma cell death via inhibition of the transient receptor potential melastatin-2 (TRPM2) cation channel. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1717. doi:10.1158/1538-7445.AM2015-1717

Abstract 5556: Enhancing response of Ganoderma lucidum (Reishi) and lapatinib in HER2+ inflammatory breast cancer cells

Abstract Inflammatory Breast Cancer (IBC) is an aggressive and unique form of breast cancer that develops rapidly. IBC, like non-IBC breast cancers, is a heterogeneous disease and can occur as any of the six molecular breast cancer subtypes. However, they are most commonly either triple negative; that is presenting with an absence of estrogen receptor (ER-), progesterone receptor (PR-) and the epidermal growth factor receptor 2 (HER2-) or ER-, PR- and HER2+ overexpressed. There is no specific IBC treatment; thus, it seems possible to find a therapeutic for this deadly disease. Our published data demonstrates that Ganoderma lucidum (Reishi) inhibits the viability of the triple negative IBC SUM149 cell line, but not of MCF10A noncancerous mammary epithelial cells. Subsequently, in this study, we aimed to investigate Reishi effects in the viability and molecular signaling of the ER-, PR-, HER2+ IBC cell lines SUM190, KPL-4 and IBC-3. Furthermore, we studied the effects of Reishi on IBC cells when treated in complement with the HER2 Tyrosine Kinase Inhibitor, lapatinib. The hypothesis for this study is that Reishi inhibits cancer cell viability and sensitizes IBC cells to lapatinib therapy and downregulates key proteins overexpressed in IBC. Herein, SUM190, KPL-4 and IBC-3 cells were treated with increasing concentrations of lapatinib and/or Reishi for 24 or 72h. The combination of lapatinib plus Reishi reduced cell viability of KPL-4 cells to 80% when treated with a concentration of 0.25 mg/mL of Reishi for 72h. Additionally, our data shows a reduction in cell viability of 70% in SUM190 cells treated with lapatinib plus Reishi for 72h. Also, Reishi and lapatinib downregulate the expression HER2 in KPL-4 cell after 72h of treatment. Our results provide evidence that Reishi inhibits cancer cell viability, highlighting the potential of Reishi as a natural anti-IBC-therapeutic. This project was sponsored by NIH/NCI #1F31 CA174307 to ISA, Title V PPOHA US Department of Education #P031M105050 to UCC/LAC, NIH/RCMI #G12 MD007583 to UCC/MMM, NIH/INBRE #5P20 GM103475 to UPR/UCC/MMM, NIH/NIMHD U54 MD008149 UH/MMM. Citation Format: Yismeilin Feliz-Mosquea, Ivette Suarez-Arroyo, Yaliz Loperena, Luis A. Cubano, Michelle M. Martinez-Montemayor. Enhancing response of Ganoderma lucidum (Reishi) and lapatinib in HER2+ inflammatory breast cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5556. doi:10.1158/1538-7445.AM2015-5556

MicroRNA-33b Inhibits Breast Cancer Metastasis by Targeting HMGA2, SALL4 and Twist1.

MicroRNAs are a class of small noncoding RNAs that regulate gene expression post-transcriptionally either by inhibiting protein translation or by causing the degradation of target mRNAs. Current evidence indicates that miR-33b is involved in the regulation of lipid metabolism, cholesterol homeostasis, glucose metabolism and several human diseases; however, whether miR-33b contributes to the pathogenesis of human cancers and participates in the regulation of self-renewal of human cancer stem cells remains unknown. Here, we report the identification of miR-33b as a negative regulator of cell stemness and metastasis in breast cancer. Compared with paired normal breast tissues, miR-33b expression is downregulated in breast tumor samples and is inversely correlated with lymph node metastatic status. Ectopic overexpression of miR-33b in highly metastatic breast cancer cells suppresses cell self-renewal, migration and invasion in vitro and inhibits lung metastasis in vivo. Conversely, miR-33b knockdown promotes the self-renewal, migration and invasion capabilities of noncancerous mammary epithelial cells. The mechanism through which miR-33b inhibits the stemness, migration and invasion of breast cancer cells is by targeting HMGA2, SALL4 and Twist1. These data indicate that miR-33b acts as an onco-suppressive microRNA in breast cancer progression by inhibiting the stemness and metastasis of breast cancer cells.

Abstract 28: Chemoprevention strategies to target abnormal imprinting in high-risk African American women who do or do not carry a BRCA1 mutation

Abstract Background: Genomic imprinting is an inherited form of parent-of-origin-specific epigenetic gene regulation that is dysregulated by poor prenatal nutrition and environmental toxins. KCNK9 encodes TASK3, a pH-regulated potassium channel membrane protein that is overexpressed in 40% of breast cancer. KCNK9 gene amplification accounts for increased expression in &amp;lt;10% of these breast cancers. Methods/Results: Here we show that KCNK9 has imprinted expression in breast tissue and provide the first identification of the KCNK9 DMR and DMR-associated open chromatin. Our studies show that loss of KCNK9 regulatory methylation and imprinted expression occurs in 75% of triple-negative breast cancer (TNBC) and loss of methylation occurs in non-cancerous tissue from high-risk women. Functional studies show that TASK3 protein regulates mitochondrial membrane potential and apoptosis-sensitivity. In live TNBC cells and non-cancerous mammary epithelial cells from high-risk women, loss of KCNK9 DMR methylation predicts increased mitochondrial membrane potential (p=0.007). African American women have a significantly higher frequency of loss of KCNK9 imprinting than do risk-matched Caucasian women (p=0.005). African American women with BRCA1 mutation have the highest frequency of loss (p&amp;lt;0.0001). Clinical Prevention: This is the first identification of the KCNK9 DMR and demonstration that loss of methylation and overexpression of TASK3 increases mitochondrial membrane potential and promotes apoptosis resistance. Low-toxicity inhibitors of TASK3 are in clinical use and can be readily re-purposed to provide cost effective chemoprevention. These inhibitors include K+ inhibitors (developed for cardiac indications) and Schwann Pepper. Our studies identify TASK3 as a “drugable”, low-toxicity target that can be readily employed as a novel target for prevention and treatment of TNBC, particularly in high-risk African American women. Citation Format: Victoria Seewaldt, David Skaar, Eric Dietze, Shraddha Desai, Randy Jirtle. Chemoprevention strategies to target abnormal imprinting in high-risk African American women who do or do not carry a BRCA1 mutation. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Susceptibility and Cancer Susceptibility Syndromes; Jan 29-Feb 1, 2014; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(23 Suppl):Abstract nr 28. doi:10.1158/1538-7445.CANSUSC14-28