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Bhardwaj, Anjana, Raniv Rojo, Zhenlin Ju, Jing Wang, and Isabelle Bedrosian. "Abstract P2-11-10: Heterogeneity of preneoplastic breast tissues drives efficacy of therapeutic agents." Cancer Research 82, no. 4_Supplement (2022): P2–11–10—P2–11–10. http://dx.doi.org/10.1158/1538-7445.sabcs21-p2-11-10.
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Abstract Introduction: Little is known about the evolution of proteomic aberrations during TNBC tumorigenesis and whether the timing of intervention for prevention and treatment during this tumorigenic progression impacts efficacy. We hypothesized that the targeted therapies for TNBC is likely to be context specific, with specific transition points where such therapy is likely to be most effective based on presence of proteomic aberrations. Methods: MCF10A cell line-based model of triple negative breast cancer progression was used. This model system comprises of non-cancer MCF10A (P), preneoplastic MCF10.AT1 and MCF10.NeoT, premalignant ductal carcinoma in situ MCF10.DCIS and invasive MCF10.CA1D cells. Global proteomic patterns were studied by performing RPPA assay. Growth inhibitory effects drugs were studied by measuring inhibition in cell proliferation by MTT assay. Results: RPPA analysis revealed a majority of protein alterations (n=48) were acquired early on during TNBC progression, specifically during normal to preneoplastic (atypia) transition, with only 6 aberrations acquired in later stages of tumorigenesis. To assess the effects of this heterogeneity on efficacy of treatment, we selected 2 aberrant pathways that were up-regulated early in the breast tumorigenesis and compared efficacy of targeted intervention to drugs without pathway selectivity. Targeting upstream oncogenic pathways with small molecule inhibitors (AKT pathway with LY294002- a PI3 kinase inhibitor and MEK pathway with MEK1/2 inhibitors PD032590 or GSK1120212) showed PI3K inhibitor to be equally effective in suppressing the cell proliferation in both preneoplastic and DCIS state, consistent with upregulation of the underlying AKT pathway aberration early in tumorigenesis. MEK pathway inhibitors were preferentially more effective in preneoplastic state than DCIS state, consistent with continued activation of MEK pathway in the DCIS state relative to preneoplastic state. Similarly, fluvastatin (a cholesterol lowering drug) was more effective in inhibiting the cell proliferation in preneoplastic cells relative to DCIS cells. However, a broad spectrum drug aspirin (an AMPK activator) that is known to have pleotropic effects showed no differential effects across the cell lines tested. Conclusions: The preponderance of proteomic alterations occur in the very earliest stages of TNBC tumorigenesis. Effectiveness of targeted drug therapies in this model of breast tumorigenesis correlated. with deregulation of the associated pathway. This implies that accounting for heterogeneity of the preneoplastic breast and stage of tumorigenic progression will be important when considering targeted strategies for prevention. Citation Format: Anjana Bhardwaj, Raniv Rojo, Zhenlin Ju, Jing Wang, Isabelle Bedrosian. Heterogeneity of preneoplastic breast tissues drives efficacy of therapeutic agents [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-11-10.
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Cando, Leslie Faye T., Zhenlin Ju, Leslie Michelle M. Dalmacio, et al. "Abstract 2360: Targeting of microRNA let-7b-5p-mediated aberrant AR signaling inhibits the growth and proliferation of preneoplastic and preinvasive stages of triple-negative breast cancer." Cancer Research 85, no. 8_Supplement_1 (2025): 2360. https://doi.org/10.1158/1538-7445.am2025-2360.
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Abstract Background: In search for targets for triple-negative breast cancer (TNBC) prevention, we analyzed molecular changes that occur early in the tumorigenic process. We hypothesize that the early loss of microRNA let-7b-5p, and subsequent upregulation of its target gene androgen receptor (AR), promotes the growth and proliferation of preneoplastic and preinvasive lesions. And while targeting AR has been studied in the therapeutic setting for breast cancer, it may also have potential use in preventing disease progression. Methods: TNBC patient samples included tumors and histologically normal tissue adjacent (<2 cm) and distant (>2 cm) from tumor. Sublines of MCF10A TNBC progression model corresponding to preneoplastic lesions MCF10.NeoT (hyperplasia) and MCF10.AT1 (atypical hyperplasia), and preinvasive tumor MCF10.DCIS (ductal carcinoma in situ) were used to study the effect of let-7b rescue and AR inhibition on proliferation, survival, or migration as measured by MTT, colony formation, and scratch assays. Let-7b gene targets were identified by cross-referencing the RNA sequencing data of MCF10A panel for upregulated genes that are predicted targets of let-7b. QPCR and RNA sequencing were used to measure AR and let-7b expression. Results: In TNBC patients, let-7b expression is reduced by 68-74% in tumors compared to adjacent (p=0.015) and distant normal tissues (p=0.048)—confirming previous results in the MCF10A model, where let-7b expression is reduced from normal-like to AT1 by 35% and to DCIS by 65% (p<0.05). Let-7b rescue results in 15-32% lower proliferation (p<0.0001), 6-9% reduction in migration (p<0.003), and a 25% (p=0.076), 66% (p<0.0001), and 55% (p=0.030) decrease in colony formation compared to scramble control in NeoT, AT1, and DCIS cells, respectively. After screening potential let-7b targets, we selected AR for validation. As expected of the inverse regulation by let-7b, AR increases in the MCF10A model, with a notable 23-fold increase in DCIS as compared to normal-like (p<0.0001), and similarly in TNBC patient tumors compared to normal tissue controls (p=0.018). Furthermore, we validated AR as a direct target with let-7b overexpression causing AR downregulation by 67% in NeoT (p=0.001), 86% in AT1 (p<0.0001), and 15% in DCIS (p=0.011). Finally, treatment with AR inhibitor enzalutamide reduced DHT-dependent AR mediated proliferation by 29-41% (p<0.0001) and colony formation by 82-95% (p<0.0001) as compared to DHT only control at a minimal concentration of 25µM in AT1 and DCIS. Conclusions: Let-7b-5p, which acts as a tumor suppressor microRNA, is lost early in tumorigenesis of TNBC, with resulting upregulation of AR. Inhibiting the AR signaling pathway impacts cellular growth and proliferation of preneoplastic and preinvasive cancer cells, suggesting a potential opportunity for TNBC prevention. Citation Format: Leslie Faye T. Cando, Zhenlin Ju, Leslie Michelle M. Dalmacio, Preethi H. Gunaratne, Jing Wang, Anjana Bhardwaj, Isabelle Bedrosian. Targeting of microRNA let-7b-5p-mediated aberrant AR signaling inhibits the growth and proliferation of preneoplastic and preinvasive stages of triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2360.
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Mullins, Stefanie R., Mansoureh Sameni, Galia Blum, Matthew Bogyo, Bonnie F. Sloane, and Kamiar Moin. "Three-dimensional cultures modeling premalignant progression of human breast epithelial cells: role of cysteine cathepsins." Biological Chemistry 393, no. 12 (2012): 1405–16. http://dx.doi.org/10.1515/hsz-2012-0252.
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Abstract The expression of the cysteine protease cathepsin B is increased in early stages of human breast cancer. To assess the potential role of cathepsin B in premalignant progression of breast epithelial cells, we employed a 3D reconstituted basement membrane overlay culture model of MCF10A human breast epithelial cells and isogenic variants that replicate the in vivo phenotypes of hyperplasia (MCF10AneoT) and atypical hyperplasia (MCF10AT1). MCF10A cells developed into polarized acinar structures with central lumens. In contrast, MCF10AneoT and MCF10AT1 cells form larger structures in which the lumens are filled with cells. CA074Me, a cell-permeable inhibitor selective for the cysteine cathepsins B and L, reduced proliferation and increased apoptosis of MCF10A, MCF10AneoT and MCF10AT1 cells in 3D culture. We detected active cysteine cathepsins in the isogenic MCF10 variants in 3D culture with GB111, a cell-permeable activity-based probe, and established differential inhibition of cathepsin B in our 3D cultures. We conclude that cathepsin B promotes proliferation and premalignant progression of breast epithelial cells. These findings are consistent with studies by others showing that deletion of cathepsin B in the transgenic MMTV-PyMT mice, a murine model that is predisposed to development of mammary cancer, reduces malignant progression.
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Hassaneen, Hamdi M., Fatma M. Saleh, Tayseer A. Abdallah, et al. "Synthesis, Cytotoxicity, Antimicrobial and Docking Simulation of Novel Pyrazolo[3,4-d]pyrimidine and pyrazolo[4,3-e][1,2,4]triazolo[3,4-c] pyrimidine Derivatives." Mini-Reviews in Medicinal Chemistry 19, no. 8 (2019): 657–70. http://dx.doi.org/10.2174/1389557518666181017162459.
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Background:Isobutyrohydrazonoyl bromide 1 was used as a precursor for the synthesis of 4-imino-3-isopropyl-1-(4-nitrophenyl)-1,4-dihydro-5H-pyrazolo[3,4-d]pyrimidin-5-amine 4, which was converted into hydrazino derivative 5 by heating with hydrazine hydrate at reflux. Hydrazino, as well as imino-amino derivatives, underwent condensation and cyclization reactions to give pyrazolo[ 3,4-d]pyrimidine and pyrazolo[4,3-e][1,2,4]triazolo[3,4-c]pyrimidine derivatives, respectively.Method:Antimicrobial studies are performed using two-gram positive bacteria and two-gram negative bacteria.Results:Data revealed that compound 9a is the most promising antibacterial agent with high efficiency (low MIC value (48 μg/ml)). The cytotoxic assay was investigated for in vitro antitumor screening against Caucasian breast adenocarcinoma MCF7, hepatocellular carcinoma HepG2 and colon carcinoma HCT-116 cell lines.Conclusion:The results are compared with doxorubicin standard anticancer drugs as well as normal cell lines like MCF10 and MCF12. Molecular docking was carried out for the highest potent compound 8c with the binding site of dihydrofolate reductase enzyme DHFR PDB:ID (1DLS).
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Merlo, G. R., F. Basolo, L. Fiore, L. Duboc, and N. E. Hynes. "p53-dependent and p53-independent activation of apoptosis in mammary epithelial cells reveals a survival function of EGF and insulin." Journal of Cell Biology 128, no. 6 (1995): 1185–96. http://dx.doi.org/10.1083/jcb.128.6.1185.
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The p53 tumor suppressor protein has been implicated as a mediator of programmed cell death (PCD). A series of nontransformed mammary epithelial cell (MEC) lines were used to correlate p53 function with activation of PCD. Treatment of MECs expressing mutant, inactive, or no p53 with DNA-damaging agents did not induce apoptosis. Upon introduction of temperature-sensitive p53 into HC11 cells, which lack wild-type (wt) p53, PCD was observed after mitomycin treatment at 32 degrees, when the ts p53 protein is in wt conformation. Thus, wt p53 mediates activation of PCD in response to mitomycin in HC11 cells. Treatment of the MCF10-A cells, which express wt p53, with various DNA-damaging agents led to nuclear accumulation of p53. Only mitomycin treatment led to an increase in the number of apoptotic nuclei. ErbB-2-transformed MCF10-A cells responded to mitomycin, cisplatin, and 5-Fl-uracil, suggesting that signaling from activated ErbB-2 enhances the cells ability to respond to DNA damage. A combination of high cell density and serum-free medium induces apoptosis in all MECs tested, irrespective of their p53 status. Under these conditions, EGF or insulin act as survival factors in preventing PCD. These data might elucidate some aspects of breast involution and tumorigenesis.
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Bhardwaj, Anjana, Zhenlin Ju, Alexander Koh, Rhea Bhala, Jing Wang, and Isabelle Bedrosian. "Abstract 5262: Avasimibe abolishes the breast cancer preventative efficacy of statin in a spontaneous mouse model of breast cancer." Cancer Research 83, no. 7_Supplement (2023): 5262. http://dx.doi.org/10.1158/1538-7445.am2023-5262.
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Abstract Background: The cholesterol biosynthesis pathway plays a central role in the normal cellular development and carcinogenesis. Relevant to breast cancer prevention, the breast epithelium in women with atypical hyperplasia has been shown to have increased in cholesterol levels and oxidative products of cholesterol. Additionally, cholesterol pathway genes such as HMGCR and HMGCS1 are known to be upregulated during progression of breast cancer in patients. We have previously shown in SV40C3TAg mice that the cholesterol lowering drug-fluvastatin reduces breast tumor incidence and burden by 50% and have noted that the efficacy of statin is reduced due to the tight regulation of the cholesterol biosynthesis pathway through multiple restorative feedback loops, thus bypassing the effect of statin blockade. Hypothesis: We hypothesize that fluvastatin efficacy can be improved by co-targeting the restorative feedback pathways that are involved in resistance to statins. Methodology: RNA seq data from statin resistant MCF10.AT1-R cell clones was compared to the statin sensitive parental MCF10.AT1 cells in order to identify pathways and targets involved in statin resistance. Genes that were also found to be upregulated in the statin non-responders mice mammary tumors relative to responders mice were tested for dual targeting. Efficacy of avasimibe, an ACAT inhibitor, to sensitize MCF10.DCIS cells to statin therapy was studied by the colony formation assay. In vivo validation of the statin + avasimibe dual therapy was performed in SV40C3TAg mice that spontaneously develop triple negative breast cancer. Results: We found ACAT1 and ACAT2 to be overexpressed in statin resistance cell clones and non-responder mouse breast tumors. ACATs are involved in cholesterol esterification that is required for storing cholesterol in lipid droplets. Cholesterol esterification in conjunction with cholesterol synthesis and export constitute key mechanisms involved in maintaining cholesterol homeostasis. Thus, we tested if avasimibe treatment potentiates the fluvastatin efficacy to inhibit colonizing ability. Avasimibe and fluvastatin combination completely abolished the ability of MCF10.DCIS cells to form colonies. Next, we tested the efficacy of combination treatment to prevent breast tumors in SV40C3TAg mice. The treatments started at the age of 6 weeks, prior to the onset of cancer and continued until 22 weeks of age. We found fluvastatin and avasimibe combination to be completely ineffective and 90% mice developed tumors. In addition, tumor burden was not reduced with dual treatment. Conclusions: We postulate that avasimibe enhanced metabolism of fluvastatin in mouse system and thus completely abolishing the chemopreventive effects of statin. Genomically derived rationale drug combinations may result in unanticipated interactions and/or ancillary effects that limit efficacy in vivo/in patients. Citation Format: Anjana Bhardwaj, Zhenlin Ju, Alexander Koh, Rhea Bhala, Jing Wang, Isabelle Bedrosian. Avasimibe abolishes the breast cancer preventative efficacy of statin in a spontaneous mouse model of breast cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5262.
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Preuss, Janina, Adam D. Richardson, Anthony Pinkerton, et al. "Identification and Characterization of Novel Human Glucose-6-Phosphate Dehydrogenase Inhibitors." Journal of Biomolecular Screening 18, no. 3 (2012): 286–97. http://dx.doi.org/10.1177/1087057112462131.
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Glucose-6-phosphate dehydrogenase (G6PD) is the key enzyme of the pentose phosphate pathway, converting glucose-6-phosphate to 6-phosphoglucono-δ-lactone with parallel reduction of NADP+. Several human diseases, including cancer, are associated with increased G6PD activity. To date, only a few G6PD inhibitors have been available. However, adverse side effects and high IC50 values hamper their use as therapeutics and basic research probes. In this study, we developed a high-throughput screening assay to identify novel human G6PD (hG6PD) inhibitors. Screening the LOPAC (Sigma-Aldrich; 1280 compounds), Spectrum (Microsource Discovery System; 1969 compounds), and DIVERSet (ChemBridge; 49 971 compounds) small-molecule compound collections revealed 139 compounds that presented ≥50% hG6PD inhibition. Hit compounds were further included in a secondary and orthogonal assay in order to identify false-positives and to determine IC50 values. The most potent hG6PD inhibitors presented IC50 values of <4 µM. Compared with the known hG6PD inhibitors dehydroepiandrosterone and 6-aminonicotinamide, the inhibitors identified in this study were 100- to 1000-fold more potent and showed different mechanisms of enzyme inhibition. One of the newly identified hG6PD inhibitors reduced viability of the mammary carcinoma cell line MCF10-AT1 (IC50 ~25 µM) more strongly than that of normal MCF10-A cells (IC50 >50 µM).
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Bhardwaj, Anjana, Alexander Koh, Rhea Bhala, et al. "Avasimibe Abolishes the Efficacy of Fluvastatin for the Prevention of Cancer in a Spontaneous Mouse Model of Breast Cancer." International Journal of Molecular Sciences 26, no. 6 (2025): 2502. https://doi.org/10.3390/ijms26062502.
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The cholesterol biosynthesis pathway is upregulated during breast cancer development and progression. Inhibition of the aberrantly upregulated cholesterol pathway by statins reduces breast tumor incidence and burden by 50% in SV40 C3(1) TAg mice, a mouse model of triple negative breast cancer. We hypothesized that fluvastatin’s preventive efficacy could be further enhanced by co-targeting the statin-induced restorative feedback pathways that tightly control the cholesterol pathway and are involved in resistance to statins. Acyl-coenzyme A: cholesterol acyltransferase (ACAT)2 is a cholesterol esterification gene that is upregulated in statin-resistant MCF10.DCIS cells, and in mammary tumors of statin-non-responsive SV40 C3(1) TAg mice. In support of this hypothesis, a combination of fluvastatin and avasimibe effectively inhibited the cell growth of statin-resistant MCF10.DCIS cells. However, this combination failed to prevent breast tumor formation in SV40 C3(1) TAg mice. Although avasimibe inhibited fluvastatin-induced ACAT2 mRNA expression in the breast tissue of the combination-treated mice, confirming that avasimibe effectively hit its target, the fluvastatin and avasimibe combination was completely ineffective in preventing breast cancer in vivo, with approximately 90% of mice developing tumors by 22 weeks, similar to the vehicle control group animals. These findings, along with avasimibe’ s known interactions with CYP450 gene family members, suggest that AVA abrogates the efficacy of fluvastatin through enhanced metabolism of fluvastatin in vivo. The findings reported in this brief communication provide a cautionary note for studies proposing the use of avasimibe in combination therapy for cancer prevention and treatment.
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Lee, Hong Jin, Andrew Wislocki, Catherine Goodman, et al. "A Novel Vitamin D Derivative Activates Bone Morphogenetic Protein Signaling in MCF10 Breast Epithelial Cells." Molecular Pharmacology 69, no. 6 (2006): 1840–48. http://dx.doi.org/10.1124/mol.105.022079.
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Brock, Ethan J., Ryan M. Jackson, Julie L. Boerner, et al. "Sprouty4 negatively regulates ERK/MAPK signaling and the transition from in situ to invasive breast ductal carcinoma." PLOS ONE 16, no. 5 (2021): e0252314. http://dx.doi.org/10.1371/journal.pone.0252314.
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Breast ductal carcinoma in situ (DCIS) is a non-obligate precursor of invasive ductal carcinoma (IDC). It is still unclear which DCIS will become invasive and which will remain indolent. Patients often receive surgery and radiotherapy, but this early intervention has not produced substantial decreases in late-stage disease. Sprouty proteins are important regulators of ERK/MAPK signaling and have been studied in various cancers. We hypothesized that Sprouty4 is an endogenous inhibitor of ERK/MAPK signaling and that its loss/reduced expression is a mechanism by which DCIS lesions progress toward IDC, including triple-negative disease. Using immunohistochemistry, we found reduced Sprouty4 expression in IDC patient samples compared to DCIS, and that ERK/MAPK phosphorylation had an inverse relationship to Sprouty4 expression. These observations were reproduced using a 3D culture model of disease progression. Knockdown of Sprouty4 in MCF10.DCIS cells increased ERK/MAPK phosphorylation as well as their invasive capability, while overexpression of Sprouty4 in MCF10.CA1d IDC cells reduced ERK/MAPK phosphorylation, invasion, and the aggressive phenotype exhibited by these cells. Immunofluorescence experiments revealed reorganization of the actin cytoskeleton and relocation of E-cadherin back to the cell surface, consistent with the restoration of adherens junctions. To determine whether these effects were due to changes in ERK/MAPK signaling, MEK1/2 was pharmacologically inhibited in IDC cells. Nanomolar concentrations of MEK162/binimetinib restored an epithelial-like phenotype and reduced pericellular proteolysis, similar to Sprouty4 overexpression. From these data we conclude that Sprouty4 acts to control ERK/MAPK signaling in DCIS, thus limiting the progression of these premalignant breast lesions.
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Weber, Ekkehard, Elena Barbulescu, Rita Medek, et al. "Cathepsin B-deficient mice as source of monoclonal anti-cathepsin B antibodies." Biological Chemistry 396, no. 3 (2015): 277–81. http://dx.doi.org/10.1515/hsz-2014-0191.
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Abstract Cathepsin B has been demonstrated to be involved in several proteolytic processes that support tumor progression and metastasis and neurodegeneration. To further clarify its role, defined monoclonal antibodies are needed. As the primary structure of human cathepsin B is almost identical to that of the mouse, cathepsin B-deficient mice were used in a novel approach for generating such antibodies, providing the chance of an increased immune response to the antigen, human cathepsin B. Thirty clones were found to produce cathepsin B-specific antibodies. Seven of these antibodies were used to detect cathepsin B in MCF10-DCIS human breast cancer cells by immunocytochemistry and immunoblotting. Five different binding sites were identified by epitope mapping giving the opportunity to combine these antibodies in oligoclonal antibody mixtures for an improved detection of cathepsin B.
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Singhal, Mitali, Jacobo Elies Gomez, Sanjit Nayak, Kirsten Riches Suman, and Amalia Ruiz Estrada. "Abstract 4386: Comparison of cytotoxicity of anthracycline based antineoplastic drugs in breast cancer." Cancer Research 83, no. 7_Supplement (2023): 4386. http://dx.doi.org/10.1158/1538-7445.am2023-4386.
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Abstract Mitoxantrone (MTX) is used for the chemotherapeutic treatment of breast cancer, but it has a dose-limiting cardiotoxicity. One of the mechanisms of MTX cytotoxicity is by inhibiting the topoisomerase II enzyme, which is crucial for maintaining cellular processes like replication. The aim of the study is to investigate if an alternative analog of MTX, named KP71 would be a suitable antineoplastic drug with reduced cardiovascular side effects. Cytotoxicity of both drugs in breast cancer cell lines MDA-MB-468, MDA-MB-231, MCF7, the non-neoplastic breast cell line MCF10, cardiac human fibroblasts (HFB), and human umbilical vein endothelial cells (HUVEC) was assessed by MTT at 96h. The IC50 values (nM) for MTX/KP71 were as shown in table below. DNA decatenation assay demonstrated topoisomerase II inhibition with values of IC50 = 2.9/12.3 µM for topoisomerase IIα, and 2.1/7.0 µM for topoisomerase IIβ for MTX/KP71 respectively. Western blotting showed DNA damage by the gradation in γH2AX expression at different concentrations of MTX and KP71 at 6 h and 24 h treatment. KP71 was found to retain the ability to inhibit the proliferation of neoplastic cell lines in vitro with a slower profile of cytotoxicity on non-neoplastic cell lines as compared to MTX. KP71 partly exerted cytotoxicity by damaging DNA and inhibiting topoisomerase II enzyme activity. Organotypic HFB-HUVEC co-cultures were used to investigate the effects of anthracycline drugs in angiogenesis. Although both drugs reduced the number of tubules (and branches) compared to control (absence of drug), there were not significant differences between MTX and KP71. MTT IC50 (nM) MTX KP71 MDA-MB-468 27 50 MDA-MB231 38 169 MCF7 63 150 MCF10 35 90 HFB 160 1177 HUVEC 56 93 Citation Format: Mitali Singhal, Jacobo Elies Gomez, Sanjit Nayak, Kirsten Riches Suman, Amalia Ruiz Estrada. Comparison of cytotoxicity of anthracycline based antineoplastic drugs in breast cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4386.
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Bayram, Nazende Nur, Gizem Tuğçe Ulu, Nusaibah Abdulsalam Abdulhadi, et al. "HER2-Specific Peptide (LTVSPWY) and Antibody (Herceptin) Targeted Core Cross-Linked Micelles for Breast Cancer: A Comparative Study." Pharmaceutics 15, no. 3 (2023): 733. http://dx.doi.org/10.3390/pharmaceutics15030733.
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This study aims to prepare a novel breast cancer-targeted micelle-based nanocarrier, which is stable in circulation, allowing intracellular drug release, and to investigate its cytotoxicity, apoptosis, and cytostatic effects, in vitro. The shell part of the micelle is composed of zwitterionic sulfobetaine ((N-3-sulfopropyl-N,N-dimethylamonium)ethyl methacrylate), while the core part is formed by another block, consisting of AEMA (2-aminoethyl methacrylamide), DEGMA (di(ethylene glycol) methyl ether methacrylate), and a vinyl-functionalized, acid-sensitive cross-linker. Following this, a targeting agent (peptide (LTVSPWY) and antibody (Herceptin®)), in varying amounts, were coupled to the micelles, and they were characterized by 1H NMR, FTIR (Fourier-transform infrared spectroscopy), Zetasizer, BCA protein assay, and fluorescence spectrophotometer. The cytotoxic, cytostatic, apoptotic, and genotoxic effects of doxorubicin-loaded micelles were investigated on SKBR-3 (human epidermal growth factor receptor 2 (HER2)-positive) and MCF10-A (HER2-negative). According to the results, peptide-carrying micelles showed a higher targeting efficiency and better cytostatic, apoptotic, and genotoxic activities than antibody-carrying and non-targeted micelles. Also, micelles masked the toxicity of naked DOX on healthy cells. In conclusion, this nanocarrier system has great potential to be used in different drug-targeting strategies, by changing targeting agents and drugs.
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McKeen Polizzotti, Lindsey, Basak Oztan, Chris S. Bjornsson, Katherine R. Shubert, Bülent Yener, and George E. Plopper. "Novel Image Analysis Approach Quantifies Morphological Characteristics of 3D Breast Culture Acini with Varying Metastatic Potentials." Journal of Biomedicine and Biotechnology 2012 (2012): 1–16. http://dx.doi.org/10.1155/2012/102036.
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Prognosis of breast cancer is primarily predicted by the histological grading of the tumor, where pathologists manually evaluate microscopic characteristics of the tissue. This labor intensive process suffers from intra- and inter-observer variations; thus, computer-aided systems that accomplish this assessment automatically are in high demand. We address this by developing an image analysis framework for the automated grading of breast cancer inin vitrothree-dimensional breast epithelial acini through the characterization of acinar structure morphology. A set of statistically significant features for the characterization of acini morphology are exploited for the automated grading of six (MCF10 series) cell line cultures mimicking three grades of breast cancer along the metastatic cascade. In addition to capturing both expected and visually differentiable changes, we quantify subtle differences that pose a challenge to assess through microscopic inspection. Our method achieves 89.0% accuracy in grading the acinar structures as nonmalignant, noninvasive carcinoma, and invasive carcinoma grades. We further demonstrate that the proposed methodology can be successfully applied for the grading ofin vivotissue samples albeit with additional constraints. These results indicate that the proposed features can be used to describe the relationship between the acini morphology and cellular function along the metastatic cascade.
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Osuala, Kingsley O., Joshua Heyza, Zhiguo Zhao, et al. "Carcinoma-Associated Fibroblasts Accelerate Growth and Invasiveness of Breast Cancer Cells in 3D Long-Term Breast Cancer Models." Cancers 16, no. 22 (2024): 3840. http://dx.doi.org/10.3390/cancers16223840.
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Background/Objectives: Carcinoma-associated fibroblasts (CAFs), a prominent cell type in the tumor microenvironment (TME), significantly contributes to cancer progression through interactions with cancer cells and other TME components. Consequently, targeting signaling pathways driven by CAFs has potential to yield new therapeutic approaches to inhibit cancer progression. However, the mechanisms underlying their long-term interactions with cancer cells in vivo remains poorly understood. Methods: To address this, we developed a three-dimensional (3D) parallel coculture model of human triple-negative breast cancer (TNBC) cells and CAFs using our innovative TAME devices. This model allowed for the analysis of TNBC paracrine interactions via their secretome over extended culture periods (at least 70 days). Results: Using TNBC cell lines (MDA-MB-231, MCF10.DCIS, and HCC70), we found that TNBC spheroids in 3D parallel cocultures with CAFs exhibited more pronounced invasive finger-like outgrowths than those in cocultures of TNBC cells and normal fibroblasts (NFs) over a period of 50–70 days. We also established that the CAF-derived secretome affects TNBC migration towards the CAF secretome region. Additionally, we observed a preferential migration of CAFs, but not NFs, toward TNBC spheroids. Conclusions: Overall, our results suggest that paracrine interactions between TNBC cells and CAFs enhance TNBC invasive phenotypes and promote reciprocal migration.
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Matsuura, Isao, Chen-Yu Lai та Keng-Nan Chiang. "Functional interaction between Smad3 and S100A4 (metastatin-1) for TGF-β-mediated cancer cell invasiveness". Biochemical Journal 426, № 3 (2010): 327–35. http://dx.doi.org/10.1042/bj20090990.
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TGF-β (transforming growth factor-β) induces a cytostatic response in most normal cell types. In cancer cells, however, it often promotes metastasis, and its high expression is correlated with poor prognosis. In the present study, we show that S100A4, a metastasis-associated protein, also called metastatin-1, can physically and functionally interact with Smad3, an important mediator of TGF-β signalling. In agreement with its known property, S100A4 binds to Smad3 in a Ca2+-dependent manner. The S100A4-binding site is located in the N-terminal region of Smad3. S100A4 can potentiate transcriptional activity of Smad3 and the related Smad2. When exogenously expressed in MCF10CA1a.cl1, an MCF10-derived breast cancer cell line, S100A4 increases TGF-β-induced MMP-9 (matrix metalloproteinase-9) expression. On the other hand, depletion of S100A4 by siRNA (small interfering RNA) from the MDA-MB231 cell line results in attenuation of MMP-9 induction by TGF-β. Consistent with these observations, S100A4 increases cell invasion ability induced by TGF-β in MCF10CA1a.cl1 cells, and depletion of the protein in MDA-MB-231 cells inhibits it. Because expression of both S100A4 and TGF-β is highly elevated in many types of malignant tumours, S100A4 and Smad3 may co-operatively increase metastatic activity of some types of cancer cells.
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Todorova, Jordana, Shazie Myashkova, Maria Petrova, Anastas Gospodinov, and Evdokia Pasheva. "The motility of breast cancer cells is stimulated by HMGB1/RAGE interaction but the truncated form lacking the C terminus has no effect." BioDiscovery 24 (September 12, 2022): e93641. https://doi.org/10.3897/biodiscovery.24.e93641.
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HMGB1/RAGE is identified as a ligand-receptor pair that plays an important role in tumorogenesis. HMGB1 and RAGE levels are higher in most human tumors and their overexpression is associated with tumor progression. The causes of breast cancer are still poorly understood. One reason might be the existence of subtypes within various cellular mechanisms as hormone-dependent and hormone -independent malignant processes. We investigated the effect of HMGB1 protein and its truncated form lacking the C terminus on the RAGE expression and cell motility of breast cancer cell lines; MCF7-noninvasive, MDA-MB-231-invasive and normal breast epithelial one MCF10. The results demonstrate that the effects of HMGB1 and HMGB1∆C through RAGE association are observed exclusively for the hormone independent MDA-MB-231 cell line. The mobility of MDA-MB-231 cells was stimulated only by the full length HMGB1. Our results suggest that HMGB1/RAGE signaling should be considered as an essential process for the development of hormone independent breast cancers with great invasive potential. The truncated form plays the role of a blocking molecule that "locks" the receptor and inactivates it. This makes the tailless molecule a promising therapeutic agent that competes for the biologically active HMGB1 ligand and prevents the downstream signaling through RAGE.
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Carter, Jennifer C., Robert A. Campbell, Jennifer A. Gibbons, Alisa S. Wolberg, and Frank C. Church. "Enhanced Cell-Associated Fibrinolytic Pathway but Not Coagulation Pathway Activity Contributes to Motility in Metastatic Breast Cancer Cells." Blood 114, no. 22 (2009): 3170. http://dx.doi.org/10.1182/blood.v114.22.3170.3170.
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Abstract Abstract 3170 Poster Board III-105 Activation of tumor cell-associated coagulation and fibrinolytic pathways occurs in many different malignant and metastatic disease processes, including breast cancer. It has been proposed that coagulation and fibrinolytic pathway activation highjack host hemostatic mechanisms and facilitate the metastatic process. However, depending on tumor cell gene expression, tumor microenvironment, and tissue of origin, it is not fully understood whether the coagulation pathway or fibrinolytic pathway, alone or together in concert, contributes to the metastatic phenotype. To characterize and compare the coagulation and fibrinolytic pathways of normal and metastatic cells, we utilized the MCF-10 family of breast cell lines. The MCF-10 family of breast cell lines was originally derived from a woman with benign fibrocystic breast disease, which led to the near normal immortalized MCF-10A cells. After transformation with T24-Ha-ras, more aggressive cell lines were derived, ultimately leading to the metastatic MCF-10CA1 cell line. Using an in vitro modified-Boyden chamber model, MCF-10CA1 cells were significantly more motile than MCF-10A cells both at baseline using BSA as the chemoattractant, and when using EGF as the chemoattractant. Thus, as predicted by properties previously reported in the literature, the metastatic MCF-10CA1 cells were substantially more motile than the near normal parental cell line, MCF-10A. Both cell types supported similar rates of factor Xa generation, plasma thrombin generation, and fibrin formation. Using laser scanning confocal microscopy, we observed that MCF-10A cells produced a stable fibrin network, whereas MCF-10CA1 cells lysed the surrounding fibrin network within 24 hours of network formation. Importantly, fibrin located proximal to (within 10 microns of) the MCF-10CA1 cell surface lysed significantly faster than fibrin located 100 microns from the surface. These results suggest that the tumor-associated fibrinolytic pathway is a key distinguishing feature between the metastatic MCF10-CA1 cells and normal MCF-10A cells. A cell-surface plasminogen activation assay showed that MCF-10CA1 cells supported substantially increased plasmin generation rates compared to MCF-10A cells, providing a mechanism for the increased fibrinolytic activity of these cells towards the fibrin network. Metastatic MCF-10CA1 cells expressed significantly increased levels of urokinase (uPA) and decreased levels of plasminogen activator inhibitor-1 (PAI-1) compared to the MCF-10A cells. Blocking uPA activity with an active-site directed-inhibitor (amiloride) decreased MCF-10CA1 cell motility to essentially the same level as MCF-10A cells. Inhibiting the phosphatidylinositol 3-kinase/Akt signaling axis of MCF-10CA1 cells with LY294002 similarly decreased cell surface plasminogen activation activity and cell motility. Collectively, these results suggest that the tumor-associated fibrinolytic pathway is a key distinguishing feature between the metastatic MCF10-CA1 cells and normal MCF-10A cells. Our results support continued investigation of urokinase inhibition, either by directly blocking uPA activity or by down-regulating uPA expression, as an attractive adjunctive therapeutic target to reduce metastatic breast cancer. Disclosures No relevant conflicts of interest to declare.
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De La Reé-Rodríguez, Sandra Carolina, María Jesús González, Ingrid Fernández, et al. "Chemical Characterization of Bioactive Compounds in Extracts and Fractions from Litopenaeus vannamei Muscle." Marine Drugs 23, no. 2 (2025): 59. https://doi.org/10.3390/md23020059.
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Marine organisms are a vital source of biologically active compounds. Organic extracts from the muscle of the Pacific white shrimp (L. vannamei) have shown antiproliferative effects on tumor cells, including breast adenocarcinoma. This study aimed to analyze these extracts’ composition and confirm their specificity for breast adenocarcinoma cells without harming normal cells. An organic chloroform extract from L. vannamei muscle was divided using a solvent partition procedure with methanol and hexane. The methanolic partition was fractionated through an open preparative liquid chromatography column to isolate compounds with biological activity, that were later tested on MDA-MB-231 (breast adenocarcinoma), and recently tested on MCF10-A (non-cancerous breast epithelial cells). Cells incubated with these fractions were assessed for viability and morphological changes using fluorescence confocal microscopy. Fractions F#13 and F#14 reduced MDA-MB-231 cancer cell viability at 100 µg/mL without affecting non-cancerous MCF-10A cells, inducing apoptosis-related changes in cancer cells. These fractions contained EPA and DHA free fatty acids, specifically F#13 contained free and esterified astaxanthin as well. The high levels of free linoleic acid 18:2 ω-6, EPA, and DHA (in a 2:1 ratio, EPA:DHA), along with free and esterified astaxanthin in F#13, significantly reduced breast adenocarcinoma cell viability, nearly to that achieved by cisplatin, a chemotherapy drug.
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Lacerda-Abreu, Marco Antonio, Thais Russo-Abrahão, Nathália Rocco-Machado, et al. "Hydrogen Peroxide Generation as an Underlying Response to High Extracellular Inorganic Phosphate (Pi) in Breast Cancer Cells." International Journal of Molecular Sciences 22, no. 18 (2021): 10096. http://dx.doi.org/10.3390/ijms221810096.
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According to the growth rate hypothesis (GRH), tumour cells have high inorganic phosphate (Pi) demands due to accelerated proliferation. Compared to healthy individuals, cancer patients present with a nearly 2.5-fold higher Pi serum concentration. In this work, we show that an increasing concentration of Pi had the opposite effect on Pi-transporters only in MDA-MB-231 when compared to other breast cell lines: MCF-7 or MCF10-A (non-tumoural breast cell line). Here, we show for the first time that high extracellular Pi concentration mediates ROS production in TNBC (MDA-MB-231). After a short-time exposure (1 h), Pi hyperpolarizes the mitochondrial membrane, increases mitochondrial ROS generation, impairs oxygen (O2) consumption and increases PKC activity. However, after 24 h Pi-exposure, the source of H2O2 seems to shift from mitochondria to an NADPH oxidase enzyme (NOX), through activation of PKC by H2O2. Exogenous-added H2O2 modulated Pi-transporters the same way as extracellular high Pi, which could be reversed by the addition of the antioxidant N-acetylcysteine (NAC). NAC was also able to abolish Pi-induced Epithelial-mesenchymal transition (EMT), migration and adhesion of MDA-MB-231. We believe that Pi transporters support part of the energy required for the metastatic processes stimulated by Pi and trigger Pi-induced H2O2 production as a signalling response to promote cell migration and adhesion.
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Mesquita, Juliana Luzía França, Aline do Carmo França Botelho, Emanuelle Carolina Honorio França, et al. "Model to study (Na+, K+)-ATPase activation in human peripheral blood mononuclear cells and MCF-10A mammary cells by the PEG-Melatonin conjugate." Caderno Pedagógico 21, no. 12 (2024): e11170. http://dx.doi.org/10.54033/cadpedv21n12-225.
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Polyethylene glycol (PEG) microsphere-modified release systems have been used for adsorption on molecules, proteins, and hormones. Melatonin (N-acetyl-5-methoxytryptamine) is a biogenic amine that regulates the activity of (Na+, K+)-ATPase. The non-tumorigenic nature of MCF10 cells can be a model for studying several biological systems, such as blood mononuclear cells. In this sense, the objective of this study was to evaluate the effects of melatonin nanofractions adsorbed to PEG microspheres on ATPase enzymes in human blood mononuclear cells and MCF-10A mammary cells. Human blood mononuclear cells and MCF-10A mammary cells (ATCC) were used. The cells were tested for (Na+, K+)-ATPase activity. It was observed that there was an increase in the enzyme in mononuclear cells that were incubated with melatonin adsorbed to the PEG microsphere. There was an increase in the enzymatic activity in MCF-10A when melatonin adsorbed to the PEG microsphere. The PEG microsphere increased the specific activity of Na+/K+-ATPase in human blood mononuclear cells. In MCF-10A mammary cells, there was no change in the presence of the PEG microsphere and a reduction in the presence of melatonin. When these cells were incubated with melatonin adsorbed to the PEG microsphere, there was an increase in the activity of Na+/K+-ATPase. The differences in enzymatic activity suggest that both cell models can serve as models to evaluate the functional activity of cells, cytotoxicity, and interactions between cells in the presence of immunomodulatory agents, thereby contributing significantly to our understanding of these processes.
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Tian, LIfeng, Xuanmao Jiao, Chenguang Wang, et al. "Abstract 3582: PPAR gamma acetylation governs mammary adenocarcinoma tumor growth via acetylated residues that determine DNA sequence-specific binding." Cancer Research 84, no. 6_Supplement (2024): 3582. http://dx.doi.org/10.1158/1538-7445.am2024-3582.
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Abstract Background: HER2/neu overexpression in breast cancer confers a lipogenic phenotype. Although the introduction of anti-Her2 therapies has led to dramatic improvements in survival, nearly all patients with metastatic Her2-positive breast cancer will progress on treatment suggesting the importance of developing coextinction approaches targeting multiple pathways. Peroxisome proliferator-activated receptor γ (PPARγ), which is expressed in a variety of malignancies, governs biological functions through transcriptional programs. PPARγ binds additional DNA cis elements associated with other transcription factors, including C/EBPs, NFκB and AP-1 proteins, to promote non-canonical signaling. Defining the mechanisms governing the selection of canonical versus non-canonical PPARγ binding sequences may provide the opportunity to design regulators with distinct functions and side effects. PPARγ1 acetylation at K268/293 participates in the regulation of adipose tissue differentiation (1), and the conserved lysine residues (K154/155) govern lipogenesis in breast cancer cells (2). Although Cre based Pparγ1 gene deletion in mammary tumor oncomice showed Pparγ1 participates in the onset and progression of ErbB2-induced mammary tumorigenesis (3), the molecular mechanisms and the post-translational modifications of PPARγ governing Pparγ1 tumorigenic function remained to be determined. Methods: Herein, we defined the role of PPARγ1 acetylation in breast cancer growth in immune-deficient mice using distinct breast cancer cell lines (MCF10-Ras, MCF10A-NeuT). Furthermore, using ChIP and ChIP-Seq we show that the acetylated residues of Pparγ1 altered preference of cis-element binding in chromatin to augment Pparγ non-canonical binding. Results: Herein, the PPARγ1 acetylated residues K154/155 were shown to be essential for the induction of transcriptional modules governing growth factor signaling, cellular apoptosis, and autophagy in mice. The K154/155 residues determined the selection of genome-wide DNA binding sites, altering the selection from canonical to non-canonical (C/EBP) DNA sequence-specific binding. The gene signature reflecting the acetylation-dependent genomic occupancy provided predictive value in patient survival outcomes. Conclusions: Endogenous Pparγ1 promotes mammary tumor onset and progression. PPARγ1 breast tumor induction involves K154/155, which governs transcriptional programs (growth, apoptosis, and autophagy) via transcriptional complex binding to canonical vs. non-canonical regulatory elements. Pparγ1 acetylation participates in ErbB2-induced breast cancer tumor growth and inflammation and may represent a relevant target for therapeutic coextinction. References: (1) Qiang L, et al. Cell 2012;150:620-322. (2) Tian L, et al. Oncotarget 2014;5:7303-153. (3) Jiao X, et al. Cancers (Basel) 2021;13. Citation Format: LIfeng Tian, Xuanmao Jiao, Chenguang Wang, Adam Ertel, Raymond Soccio, Eric R. Chen, Balázs Győrffy, Gabriele Di Sante, Zhijiu Zhong, Sankar Addya, Peter A. McCue, Andrew V. Kossenkov, Joanna Achinger-Kawecka, Susan Clark, Richard G. Pestell. PPAR gamma acetylation governs mammary adenocarcinoma tumor growth via acetylated residues that determine DNA sequence-specific binding [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3582.
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Hannafon, Bethany, Samrita Dogra, Sugantha Priya Elayapillai, et al. "Abstract B012: Exosome microRNAs are progressively altered and contribute to invasive progression of ductal carcinoma in situ." Cancer Prevention Research 15, no. 12_Supplement_1 (2022): B012. http://dx.doi.org/10.1158/1940-6215.dcis22-b012.
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Abstract Ductal carcinoma in situ (DCIS) is a benign breast condition that increases the risk of invasive breast cancer (IBC). Clinically, not all DCIS will progress to IBC and the major drivers of progression are not well understood. The role of small extracellular vesicles, also known as exosomes, is well described in advanced cancers, however, little is known about their role in the invasive progression of in situ breast disease. In this study, we sought to evaluate the role of exosomes and their RNA contents in the progression of DCIS, using the MCF10 isogenic series, in vitro assays and the mouse intraductal model of breast cancer progression. We observed a significant increase of exosome loading of microRNAs (miRNAs) between normal and benign states that increased with each stage. We demonstrate that pro-invasive programs were attenuated when overall exosome and miRNA secretion was inhibited by Rab27A and Dicer knockdown, respectively. Comparisons of exosome miRNA expression changes revealed the greatest differential expression between in situ and invasive cells, with miR-205 being the most significantly different, suggesting a mechanistic role for exosome miRNAs in invasive progression. Modulation of miR-205 levels affected epithelial to mesenchymal transition and the invasive programs in vitro. Progression of DCIS in vivo resulted in increased levels of specific circulating exosome miRNAs, while inhibition of exosome secretion (Rab27A knockdown) attenuated invasive progression and reduced circulating levels of exosome miRNAs. These observations suggest that exosome miRNAs are differentially loaded and expressed, and promote the invasive progression of DCIS and that circulating exosome miRNAs are a potential source of biomarkers of the DCIS to invasive transition. Citation Format: Bethany Hannafon, Samrita Dogra, Sugantha Priya Elayapillai, James Lausen, Matthew Bruns, Fariba Behbod, Amy Gin, Chao Xu, Roy Zhang, Wei-Qun Ding. Exosome microRNAs are progressively altered and contribute to invasive progression of ductal carcinoma in situ [abstract]. In: Proceedings of the AACR Special Conference on Rethinking DCIS: An Opportunity for Prevention?; 2022 Sep 8-11; Philadelphia, PA. Philadelphia (PA): AACR; Can Prev Res 2022;15(12 Suppl_1): Abstract nr B012.
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Chornoguz, Olesya, Alexei Gapeev, Michael C. O'Neill, and Suzanne Ostrand-Rosenberg. "Major Histocompatibility Complex Class II+ Invariant Chain Negative Breast Cancer Cells Present Unique Peptides that Activate Tumor-specific T Cells from Breast Cancer Patients." Molecular & Cellular Proteomics 11, no. 11 (2012): 1457–67. http://dx.doi.org/10.1074/mcp.m112.019232.
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The major histocompatibility complex (MHC) class II-associated Invariant chain (Ii) is present in professional antigen presenting cells where it regulates peptide loading onto MHC class II molecules and the peptidome presented to CD4+ T lymphocytes. Because Ii prevents peptide loading in neutral subcellular compartments, we reasoned that Ii− cells may present peptides not presented by Ii+ cells. Based on the hypothesis that patients are tolerant to MHC II-restricted tumor peptides presented by Ii+ cells, but will not be tolerant to novel peptides presented by Ii− cells, we generated MHC II vaccines to activate cancer patients' T cells. The vaccines are Ii− tumor cells expressing syngeneic HLA-DR and the costimulatory molecule CD80. We used liquid chromatography coupled with mass spectrometry to sequence MHC II-restricted peptides from Ii+ and Ii− MCF10 human breast cancer cells transfected with HLA-DR7 or the MHC Class II transactivator CIITA to determine if Ii− cells present novel peptides. Ii expression was induced in the HLA-DR7 transfectants by transfection of Ii, and inhibited in the CIITA transfectants by RNA interference. Peptides were analyzed and binding affinity predicted by artificial neural net analysis. HLA-DR7-restricted peptides from Ii− and Ii+ cells do not differ in size or in subcellular location of their source proteins; however, a subset of HLA-DR7-restricted peptides of Ii− cells are not presented by Ii+ cells, and are derived from source proteins not used by Ii+ cells. Peptides from Ii− cells with the highest predicted HLA-DR7 binding affinity were synthesized, and activated tumor-specific HLA-DR7+ human T cells from healthy donors and breast cancer patients, demonstrating that the MS-identified peptides are bonafide tumor antigens. These results demonstrate that Ii regulates the repertoire of tumor peptides presented by MHC class II+ breast cancer cells and identify novel immunogenic MHC II-restricted peptides that are potential therapeutic reagents for cancer patients.
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Cottone, Gannon, Mariana Bustamante Eduardo, Shivangi Yadav, Seema Khan, and Susan Clare. "Abstract P6-11-08: Non-transformed breast epithelial cells show neural-like gene signature after lipid exposure." Cancer Research 83, no. 5_Supplement (2023): P6–11–08—P6–11–08. http://dx.doi.org/10.1158/1538-7445.sabcs22-p6-11-08.
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Abstract Introduction: The identification of women specifically at risk for estrogen receptor negative breast cancer (ER-BC) and the targeted treatment of this disease are significantly unmet clinical needs. To that end, we analyzed the gene expression profiles of epithelial cells from the contralateral unaffected breasts (CUBs) of BC patients and identified a lipid metabolism gene signature, which was enriched in the CUBs of women with ER-negative BC (PMID: 28263391). Subsequent experiment revealed that exposure of non-transformed breast epithelial cells to lipid results in significant changes gene expression, chromatin accessibility and histone posttranslational modifications (PMID: 35508495). Several of the upregulated genes are hallmarks of the various fates of vagal neural crest: Neural, neurogenic and mesenchymal lineages. We hypothesize that lipid exposure imparts a survival advantage of stem-like cells, that lipid-induced epigenetic changes lead to a neural crest-like transcription signature and that these genes are not expressed normally in the breast. Methods: MCF10A cells were exposed to vehicle or octanoic acid (OA) for 24 hours. Gene expression was assayed by RNA-seq and OA responsive genes were identified (PMID: 35508495). Single-cell RNA sequencing (scRNA-seq) data from 14 human reduction mammoplasties (RM) was obtained from a publicly accessible data set (PMID: 34031589). The scRNA data was clustered and identified by unsupervised clustering (Seurat, v3.4.1) using cell-type markers curated using Supplementary table 2 from (PMID: 34031589). The bulk RNA-seq data from the OA treated cells was deconvoluted to cell-lineages using Bisque. The most significant upregulated VNC neural/neuronal/mesenchymal genes from the gene expression analysis were then plotted on the lineage clusters using FeaturePlot to determine if these markers are found in the normal breast epithelium, or other cell lineages. The plots were then filtered and re-clustered to look at basal-luminal cell types only. We utilized a second resource, a web-application for snATAC-seq data from various stages of mouse mammary development developed by the Wahl lab (https://wahl-lab-salk.shinyapps.io/Mammary_snATAC/), to query these same genes. Results: Deconvolution of the bulk RNA-sequencing data revealed a transition to a pericyte transcription program following exposure to OA. Nerve growth factor (NGF) was found to be expressed in pericytes while nerve growth factor receptor (NGFR) was found within the basal epithelial cell lineage. Genes overexpressed in the VNC neural cluster and overexpressed in the OA-exposed MCF10 cells, PPP1R1C (2.39x, adj p=1.6E-5), FOXD3 (6.7x, adj p=6.7E-10), DIO3 (5.9x, adj p=3.9E-6) and MOXD3 (4.2x, adj p=1.5E-23) all evidence little to no expression in the normal breast but are observed in murine fetal mammary stem cells. Schwann cell precursor (SCP) markers, CDH19 and ROPN1, significantly upregulated in OA treated cells: 5.4x and 1.6x, respectively, exhibited low expression in luminal progenitors but in the mouse were observed in the mammary stroma. CDH19, a gene exclusive to SCPs, is expressed in stroma following murine birth. PRRX1, a key regulator of the VNC mesenchymal cell fate cluster, is 9.3-fold (p adj=4.9E-49) overexpressed in the OA treated cells, expressed strongly in pericytes and stroma and to a lesser extent in basal epithelial cells of the normal human breast and stroma of the murine mammary gland. Conclusions: Treatment of non-transformed mammary epithelial cells with lipid, specifically OA, shows significant upregulation of multiple VNC genes associated with both neural and mesenchymal fate. scRNA-seq from RM patients reveals that many of these same markers are either found in non-epithelial cell clusters or are found with low expression in luminal mammary lineages (both progenitors and mature). Citation Format: Gannon Cottone, Mariana Bustamante Eduardo, Shivangi Yadav, Seema Khan, Susan Clare. Non-transformed breast epithelial cells show neural-like gene signature after lipid exposure [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-11-08.
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Giorgio, Eros Di, Valentina Cutano, Martina Minisini, Vanessa Tolotto, Emiliano Dalla, and Claudio Brancolini. "A regulative epigenetic circuit supervised by HDAC7 represses IGFBP6 and IGFBP7 expression to sustain mammary stemness." Epigenomics 13, no. 9 (2021): 683–98. http://dx.doi.org/10.2217/epi-2020-0347.
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Background: In the breast, the pleiotropic epigenetic regulator HDAC7 can influence stemness. Materials & Methods: The authors used MCF10 cells knocked-out for HDAC7 to explore the contribution of HDAC7 to IGF1 signaling. Results: HDAC7 buffers H3K27ac levels at the IGFBP6 and IGFBP7 genomic loci and influences their expression. In this manner, HDAC7 can tune IGF1 signaling to sustain stemness. In HDAC7 knocked-out cells, RXRA promotes the upregulation of IGFBP6/7 mRNAs. By contrast, HDAC7 increases FABP5 expression, possibly through repression of miR-218. High levels of FABP5 can reduce the delivery of all-trans-retinoic acid to RXRA. Accordingly, the silencing of FABP5 increases IGFBP6 and IGFBP7 expression and reduces mammosphere generation. Conclusion: The authors propose that HDAC7 controls the uptake of all-trans-retinoic acid, thus influencing RXRA activity and IGF1 signaling.
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Nanua, Suparna, and Fayth K. Yoshimura. "Mink Epithelial Cell Killing by Pathogenic Murine Leukemia Viruses Involves Endoplasmic Reticulum Stress." Journal of Virology 78, no. 21 (2004): 12071–74. http://dx.doi.org/10.1128/jvi.78.21.12071-12074.2004.
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ABSTRACT We previously demonstrated that mink cells undergo apoptosis after MCF13 murine leukemia virus (MLV) infection. In this study, we observed that virus-infected mink epithelial cells had significantly larger amounts of steady-state levels of MCF13 MLV envelope precursor protein (gPr80 env ) than did Mus dunni fibroblasts, which are resistant to virus-induced cytopathicity. Infection of mink cells with the noncytopathic NZB-9 MLV did not result in the accumulation of gPr80 env . MCF13 MLV infection of mink cells produced low cell surface expression of envelope glycoprotein and less efficient spread of infectious virus. Western blot analysis of mink epithelial cells infected with MCF13 MLV showed an increase in GRP78/BiP, which was not observed for either mink cells infected with NZB-9 MLV or M. dunni fibroblasts infected with MCF13 MLV. MCF13 MLV infection of mink cells also resulted in a significant upregulation of CHOP/GADD153. These results indicate that the accumulation of MCF13 MLV gPr80 env triggers endoplasmic reticulum stress, which may mediate apoptosis in mink epithelial cells.
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Takeno, Takayoshi, Takuya Hasegawa, Hiroki Hasegawa, et al. "MicroRNA-205-5p inhibits three-dimensional spheroid proliferation of ErbB2-overexpressing breast epithelial cells through direct targeting of CLCN3." PeerJ 7 (October 8, 2019): e7799. http://dx.doi.org/10.7717/peerj.7799.
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We previously reported that microRNA-205-5p (miR-205-5p) is significantly decreased in the ErbB2-overexpressing breast epithelial cell line MCF10A-ErbB2 compared with control cells. In this study, we identified a direct target of miR-205-5p, chloride voltage-gated channel 3 (CLCN3). CLCN3 expression was induced by ErbB2 overexpression; this induced expression was then reduced to control levels by the transfection of the miR-205-5p precursor. In RNA-binding protein immunoprecipitation with Ago1/2/3 antibody, CLCN3 was significantly enriched in 293T embryonic kidney cells with miR-205-5p mimic transfection compared with negative control mimic transfection. In luciferase reporter assays using CLCN3 3′-UTR constructs, the miR-205-5p mimic significantly decreased reporter activity of both wild-type and partial mutant constructs in MCF10A-ErbB2 cells. In contrast, no inhibitory effects of the miR-205-5p mimic were detected using the complete mutant constructs. Since miR-205-5p expression in exosomes derived from MCF10A-neo cells was substantially higher than in exosomes derived from MCF10A-ErbB2 cells, we next investigated whether an exosome-mediated miR-205-5p transfer could control CLCN3 expression. To this end, exosomal miR-205-5p derived from MCF10A-neo cells was functionally transferred to MCF10A-ErbB2 cells, which served to decrease the expression of CLCN3. To assess the roles of CLCN3 in breast cancer, we next performed three-dimensional (3D) spheroid proliferation analyses using MCF10A-ErbB2 cells treated with MCF10A-neo-derived exosomes or CLCN3 shRNA stably expressing SKBR3 and MDA-MB-453 breast cancer cells. Our results showed that both treatment with MCF10A-neo-derived exosome and CLCN3 shRNA expression suppressed 3D spheroid proliferation. Collectively, these novel findings suggest that CLCN3 may be a novel direct target of miR-205-5p and this CLCN3/miR-205-5p interaction may serve a pivotal role in regulating breast cancer cellular proliferation under physiological conditions.
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Risha, Y., V. Susevski, N. Hüttmann, S. Poolsup, Z. Minic, and M. V. Berezovski. "Proteome of breast cancer derived microvesicles." Siberian Medical Review, no. 2 (2021): 68–71. http://dx.doi.org/10.20333/25000136-2021-2-68-71.
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The aim of the research. To examine the proteomic profi le of breast cancer exosomes. Material and methods. Cell lines used for this study were MDA-MB-231 female epithelial breast cancer cells (ATCC HTB-26) and MCF10A non-tumorigenic epithelial breast tissue cells. MVs were isolated using diff erential ultracentrifugation. Samples were lysed, reduced, alkylated, digested, and analyzed by an Orbitrap Fusion mass spectrometer. MS raw fi les were analyzed using MaxQuant version 1.6.12.0. Peptides were searched against the human UniProt FASTA database using the Andromeda search engine, integrated into MaxQuant. Results. MVs derived from MCF10A and MDA-MB-231 cell lines were analyzed, and 1427 and 547 proteins were identifi ed in the MDA-MB-231 and MCF10A-derived MVs, respectively. In total, 455 proteins were common to both MDA-MB-231 and MCF10A MVs. MVs derived from MCF10A and MDAMB-231 cell lines were analyzed, and 1427 and 547 proteins were identifi ed in the MDA-MB-231 and MCF10A-derived MVs, respectively. In total, 455 proteins were common to both MDA-MB-231 and MCF10A MVs. Th e unique MDA-MB-231 MV proteins were searched against the DisGeNET human diseases database. Out of 972 MDA-MB-231 MV proteins, 112 were cancer-related while 32 were specifi cally associated with BC. In the MDA-MB-231 MV proteome, 23 Wnt signaling pathway proteins were identifi ed based on their GO biological process. Proteomic analysis identifi ed enzymes OAT, TALDO1, and BLMH were only in MVs from metastatic MDA-MB-231 cell line. The specific activity of OAT and TALDO1 was higher in MV fractions of MDA-MB-231 in comparison to the non-cancerous MCF10A cell line-derived MVs. Th ese fi ndings might suggest that these enzymes might play a role in BC. In our present study, we found that some enzymes identifi ed from MV fractions were already proposed to play a role in cancer therapy as therapeutic targets (OAT, TALDO1) and resistance against chemotherapy agents (BLMH).
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Brew, Tom, Nicola Bougen-Zhukov, Wilson Mitchell, et al. "Loss of E-Cadherin Leads to Druggable Vulnerabilities in Sphingolipid Metabolism and Vesicle Trafficking." Cancers 14, no. 1 (2021): 102. http://dx.doi.org/10.3390/cancers14010102.
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Germline inactivating variants of CDH1 are causative of hereditary diffuse gastric cancer (HDGC), a cancer syndrome characterized by an increased risk of both diffuse gastric cancer and lobular breast cancer. Because loss of function mutations are difficult to target therapeutically, we have taken a synthetic lethal approach to identify targetable vulnerabilities in CDH1-null cells. We have previously observed that CDH1-null MCF10A cells exhibit a reduced rate of endocytosis relative to wildtype MCF10A cells. To determine whether this deficiency is associated with wider vulnerabilities in vesicle trafficking, we screened isogenic MCF10A cell lines with known inhibitors of autophagy, endocytosis, and sphingolipid metabolism. Relative to wildtype MCF10A cells, CDH1−/− MCF10A cells showed significantly greater sensitivity to several drugs targeting these processes, including the autophagy inhibitor chloroquine, the endocytosis inhibitors chlorpromazine and PP1, and the sphingosine kinase 1 inhibitor PF-543. Synthetic lethality was confirmed in both gastric and mammary organoid models of CDH1 loss, derived from CD44-Cre/Cdh1fl/fl/tdTomato mice. Collectively, these results suggest that both sphingolipid metabolism and vesicle trafficking represent previously unrecognised druggable vulnerabilities in CDH1-null cells and may lead to the development of new therapies for HDGC.
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Yoshimura, Fayth K., Tao Wang, and Suparna Nanua. "Mink Cell Focus-Forming Murine Leukemia Virus Killing of Mink Cells Involves Apoptosis and Superinfection." Journal of Virology 75, no. 13 (2001): 6007–15. http://dx.doi.org/10.1128/jvi.75.13.6007-6015.2001.
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ABSTRACT Induction of apoptosis by different types of pathogenic retroviruses is an important step in disease development. We have observed that infection of thymic lymphocytes by the mink cell focus-forming murine leukemia virus (MCF MLV) during the preleukemic period resulted in an enhancement of apoptosis of these cells. To further study the ability of MCF MLVs to induce apoptosis and the role of this process in viral pathogenesis, we have developed an in vitro system of virus-induced apoptosis. MCF13 MLV infection of mink epithelial cells resulted in the production of cytopathic foci. In contrast, infection of mink cells with the 4070A amphotropic MLV did not produce any cytopathic effects. Staining of MCF13 MLV-infected cells with propidium iodide and annexin V-fluorescein isothiocyanate indicated that virus-induced cell death was due to apoptosis. At 6 days postinfection, the percentage of apoptotic MCF13 MLV-infected cells was 27% compared with 2 to 3% for mock- or amphotropic MLV-infected cells, representing a 9- to 14-fold difference. Assays for caspase-3 activation confirmed the detection by flow cytometry of apoptosis of MCF13 MLV-infected cells. Large amounts of unintegrated linear viral DNA were detectable by Southern blot analysis during the acute phase of infection, which indicated that MCF13 MLV is able to superinfect mink cells. Unintegrated viral DNA of only the linear form was detectable in thymic lymphocytes isolated from MCF13 MLV-inoculated mice during the preleukemic period. These results indicated that the ability of MCF13 MLV to induce apoptosis is correlated with its ability to superinfect cells and that this occurs as an early step in thymic lymphoma development.
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Vydra, Natalia, Agnieszka Toma-Jonik, Patryk Janus, et al. "An Increase in HSF1 Expression Directs Human Mammary Epithelial Cells toward a Mesenchymal Phenotype." Cancers 15, no. 20 (2023): 4965. http://dx.doi.org/10.3390/cancers15204965.
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HSF1 is a well-known heat shock protein expression regulator in response to stress. It also regulates processes important for growth, development or tumorigenesis. We studied the HSF1 influence on the phenotype of non-tumorigenic human mammary epithelial (MCF10A and MCF12A) and several triple-negative breast cancer cell lines. MCF10A and MCF12A differ in terms of HSF1 levels, morphology, growth in Matrigel, expression of epithelial (CDH1) and mesenchymal (VIM) markers (MCF10A are epithelial cells; MCF12A resemble mesenchymal cells). HSF1 down-regulation led to a reduced proliferation rate and spheroid formation in Matrigel by MCF10A cells. However, it did not affect MCF12A proliferation but led to CDH1 up-regulation and the formation of better organized spheroids. HSF1 overexpression in MCF10A resulted in reduced CDH1 and increased VIM expression and the acquisition of elongated fibroblast-like morphology. The above-mentioned results suggest that elevated levels of HSF1 may direct mammary epithelial cells toward a mesenchymal phenotype, while a lowering of HSF1 could reverse the mesenchymal phenotype to an epithelial one. Therefore, HSF1 may be involved in the remodeling of mammary gland architecture over the female lifetime. Moreover, HSF1 levels positively correlated with the invasive phenotype of triple-negative breast cancer cells, and their growth was inhibited by the HSF1 inhibitor DTHIB.
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Martin, Stuart S., and Philip Leder. "Human MCF10A Mammary Epithelial Cells Undergo Apoptosis following Actin Depolymerization That Is Independent of Attachment and Rescued by Bcl-2." Molecular and Cellular Biology 21, no. 19 (2001): 6529–36. http://dx.doi.org/10.1128/mcb.21.19.6529-6536.2001.
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ABSTRACT Many tumor cells are impaired in adhesion-regulated apoptosis, which contributes to their metastatic potential. However, suppression of this apoptotic pathway in untransformed cells is not mediated only by adhesion to the extracellular matrix but also through the resulting ability to spread and adopt a distinct morphology. Since cell spreading is dependent on the integrity of the actin microfilament cytoskeleton, we sought to determine if actin depolymerization was sufficient to induce apoptosis, even in the presence of continuous attachment. For this study, we used a human mammary epithelial cell line (MCF10A), which is immortalized but remains adhesion dependent for survival. Treatment of MCF10A cells with latrunculin-A (LA), an inhibitor of actin polymerization, rapidly led to disruption of the actin cytoskeleton and caused cell rounding but preserved attachment. Initiation of apoptosis in LA-treated MCF10A cells was detected by mitochondrial localization of the Bax apoptotic protein, which was prevented by overexpression of Bcl-2. DNA fragmentation and poly(ADP-ribose) polymerase (PARP) cleavage in LA-treated MCF10A cells indicated progression to the execution phase of apoptosis. The MDA-MB-453 cell line, which was derived from a metastatic human mammary tumor, was resistant to PARP cleavage and loss of viability in response to actin depolymerization. Stable overexpression of Bcl-2 in the untransformed MCF10A cells was able to recapitulate the resistance to apoptosis found in the tumor cell line. We demonstrate that inhibition of actin polymerization is sufficient to stimulate apoptosis in attached MCF10A cells, and we present a novel role for Bcl-2 in cell death induced by direct disruption of the actin cytoskeleton.
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Nanua, Suparna, and Fayth K. Yoshimura. "Differential Cell Killing by Lymphomagenic Murine Leukemia Viruses Occurs Independently of p53 Activation and Mitochondrial Damage." Journal of Virology 78, no. 10 (2004): 5088–96. http://dx.doi.org/10.1128/jvi.78.10.5088-5096.2004.
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ABSTRACT Upon inoculation into AKR mice, mink cell focus-forming murine leukemia virus (MCF MLV) accelerates thymic lymphoma formation. During the preleukemic phase of disease, we observed the induction of apoptosis in thymic lymphocytes. A similar induction of apoptosis was observed for cultured mink epithelial cells after MCF13 MLV infection. In this study, the relevance of viral pathogenicity to cell killing was determined by testing the susceptibility of various cell types from different species to lymphomagenic MLVs. We observed that the cytopathic effect of lymphomagenic MLVs was restricted to mink cells. Southern blot analysis of MLV-infected cells revealed an accumulation of the linear form of unintegrated viral DNA, particularly in mink cells after MCF13 MLV infection. Thus, a strong correlation was observed between viral superinfection, which results in the accumulation of high levels of unintegrated viral DNA, and cell killing. Immunoblot analysis for MCF13 MLV-infected mink epithelial cells did not show a significant change in total p53 levels or its phosphorylated form at Ser-15 compared with that in mock-treated cells. Moreover, a time course analysis for mink epithelial cells infected with MCF13 MLV did not reveal mitochondrial depolarization or a significant change in Bax levels. These results demonstrate that MCF13 MLV induces apoptosis preferentially in cells in which superinfection occurs, and the mechanism involved is independent of p53 activation and mitochondrial damage.
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Huang, J., J. D. Hardy, Y. Sun, and J. E. Shively. "Essential role of biliary glycoprotein (CD66a) in morphogenesis of the human mammary epithelial cell line MCF10F." Journal of Cell Science 112, no. 23 (1999): 4193–205. http://dx.doi.org/10.1242/jcs.112.23.4193.
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Normal mammary epithelial cells express the cell surface protein biliary glycoprotein (BGP or CD66a) in a polarized manner, suggesting that this protein may play a role in the formation of mammary acini. In order to test this hypothesis, we interrupted the expression of BGP in the mammary epithelial line MCF10F when cultured in or on Matrigel, a source of extracellular matrix (ECM). When analyzed by immunofluorescence confocal microscopy, the BGP staining is confined to the lumenal surface and colocalizes with actin. Sequential scanning electron microscopy demonstrates that the MCF10F cells migrate to form clusters, followed by apoptotic cell death within the center, resulting in lumen formation. Transmission electron micrographs reveal the presence of tight junctions and desmosomes between the cells, microvilli along the lumenal surface, and typical apoptotic bodies within the lumen. When the MCF10F cells are transfected with the BGP antisense gene and grown in Matrigel, they exhibit reduced acini formation (12% and 20%) compared to untransfected cells (52%) or to cells transfected with vector only (62%). Acini formation is also significantly reduced when MCF10F cells grown in Matrigel are treated with anti-BGP antibody (18% at 100 microgram/ml), or recombinant soluble BGP (18% at 0.4 microM). In contrast, the BGP-negative MCF7 breast tumor cell line, which does not form acini when grown in matrigel, exhibits &gt;60% cell death with the occasional formation of acini, when transfected with the BGP sense gene and grown in Matrigel. These results support the hypothesis that BGP plays a role in the normal differentiation program of mammary epithelial cells, indicating that its expression is essential to the formation of the lumen. Furthermore, and as shown by others, the differentiation program depends on the presence of ECM. The lack of expression of BGP in the MCF7 breast cancer cell line suggests that the downregulation of BGP expression confers a growth advantage to these cells in ECM. In addition, we found that the MCF10F cells could be separated into a BGP-positive epithelial fraction (MCF10F-e), and a BGP-negative myoepithelial fraction (MCF10F-m). When the myoepithelial cell-enriched fraction is grown on Matrigel, web-like structures are formed. These cells have a typical spindle shape cell morphology and express keratin, alpha-smooth muscle actin and vimentin, markers of the myoepithelial cell phenotype. When MCF10F-m cells are treated with IFNgamma, they express CEA (carcinoembryonic antigen) but not BGP. Since breast carcinomas, especially in situ carcinomas, express CEA, this finding may suggest a heretofore unappreciated relationship between myoepithelial cells and breast cancer.
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McGuinness, Charlotte B., Megan A. Wilson, Margaret V. Leonard, Maria Ouzounova, Hasan Korkaya, and Austin Y. Shull. "Abstract 1740: Histone proteomic profiling of EMT-transformed MCF10A breast cells demonstrates a loss in novel histone arginine methylation sites via dual p53 and PTEN deletion." Cancer Research 84, no. 6_Supplement (2024): 1740. http://dx.doi.org/10.1158/1538-7445.am2024-1740.
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Abstract Metastatic potential of basal-like breast cancers typically is initiated by genetic alterations that lead to a process known as epithelial-mesenchymal transition (EMT). However, much is currently not understood regarding the role of epigenetic modifications that lead to invasive characteristics and EMT phenotype of metastatic breast cancers. Based on the previous notion connecting epigenetic changes to breast cancer metastasis, we performed DIA-based mass spectrometry of isolated histones from an isogenic panel of MCF10A breast cell lines where the tumor suppressor genes TP53 and PTEN were silenced and induced EMT. With this approach, we determined which histone modifications were differentially enriched in the non-EMT and EMT-induced MCF10A cell lines. From approximately 72 histone modifications identified and annotated from our mass spectrometry results, we were able to identify 5 histone events that were differentially enriched in our MCF10A cell line panel. Two events of note were histone H3 lysine-14 acetylation (H3K14ac) significantly increasing and histone H4 arginine 55 dimethylation (H4R55me2) significantly decreasing in our EMT-transformed MCF10A p53-/PTEN- cell lines when compared to the parental, non-tumorigenic MCF10A cell line, showing these events are differentially affected during the EMT process in breast cancer cells. Furthermore, significant arginine demethylation of H4R55me2 & H3.1R83me in the EMT-transformed MCF10A p53-/PTEN- cell lines corresponded with JMJD6, an established histone arginine demethylase, being overexpressed in basal-like breast cancer cell lines as well as basal-like breast cancer patients from The Cancer Genome Atlas (TCGA) and METABRIC datasets. Based on histone proteomic profiling of our isogenic cell line model, the loss of specific histone arginine methylation events corresponding with JMJD6 overexpression could highlight the potential for a targetable epigenetic mechanism in breast cancer metastasis. Citation Format: Charlotte B. McGuinness, Megan A. Wilson, Margaret V. Leonard, Maria Ouzounova, Hasan Korkaya, Austin Y. Shull. Histone proteomic profiling of EMT-transformed MCF10A breast cells demonstrates a loss in novel histone arginine methylation sites via dual p53 and PTEN deletion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1740.
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Machida, Yuichi J., Yuefeng Chen, Yuka Machida, Ankit Malhotra, Sukumar Sarkar, and Anindya Dutta. "Targeted Comparative RNA Interference Analysis Reveals Differential Requirement of Genes Essential for Cell Proliferation." Molecular Biology of the Cell 17, no. 11 (2006): 4837–45. http://dx.doi.org/10.1091/mbc.e06-04-0340.
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Differences in the genetic and epigenetic make up of cell lines have been very useful for dissecting the roles of specific genes in the biology of a cell. Targeted comparative RNAi (TARCOR) analysis uses high throughput RNA interference (RNAi) against a targeted gene set and rigorous quantitation of the phenotype to identify genes with a differential requirement for proliferation between cell lines of different genetic backgrounds. To demonstrate the utility of such an analysis, we examined 257 growth-regulated genes in parallel in a breast epithelial cell line, MCF10A, and a prostate cancer cell line, PC3. Depletion of an unexpectedly high number of genes (25%) differentially affected proliferation of the two cell lines. Knockdown of many genes that spare PC3 (p53−) but inhibit MCF10A (p53+) proliferation induces p53 in MCF10A cells. EBNA1BP2, involved in ribosome biogenesis, is an example of such a gene, with its depletion arresting MCF10A at G1/S in a p53-dependent manner. TARCOR is thus useful for identifying cell type–specific genes and pathways involved in proliferation and also for exploring the heterogeneity of cell lines. In particular, our data emphasize the importance of considering the genetic status, when performing siRNA screens in mammalian cells.
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Zhong, Cuiling, Michael S. Kinch, and Keith Burridge. "Rho-stimulated Contractility Contributes to the Fibroblastic Phenotype of Ras-transformed Epithelial Cells." Molecular Biology of the Cell 8, no. 11 (1997): 2329–44. http://dx.doi.org/10.1091/mbc.8.11.2329.
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Oncogenic transformation of cells alters their morphology, cytoskeletal organization, and adhesive interactions. When the mammary epithelial cell line MCF10A is transformed by activated H-Ras, the cells display a mesenchymal/fibroblastic morphology with decreased cell–cell junctions but increased focal adhesions and stress fibers. We have investigated whether the transformed phenotype is due to Rho activation. The Ras-transformed MCF10A cells have elevated levels of myosin light chain phosphorylation and are more contractile than their normal counterparts, consistent with the activation of Rho. Furthermore, inhibitors of contractility restore a more normal epithelial phenotype to the Ras-transformed MCF10A cells. However, inhibiting Rho by microinjection of C3 exotransferase or dominant negative RhoA only partially restores the normal phenotype, in that it fails to restore normal junctional organization. This result prompted us to examine the effect that inhibiting Rho would have on the junctions of normal MCF10A cells. We have found that inhibiting Rho by C3 microinjection leads to a disruption of E-cadherin cytoskeletal links in adherens junctions and blocks the assembly of new adherens junctions. The introduction of constitutively active Rho into normal MCF10A cells did not mimic the Ras-transformed phenotype. Thus, these results lead us to conclude that some, but not all, characteristics of Ras-transformed epithelial cells are due to activated Rho. Whereas Rho is needed for the assembly of adherens junctions, high levels of activated Rho in Ras-transformed cells contribute to their altered cytoskeletal organization. However, additional events triggered by Ras must also be required for the disruption of adherens junctions and the full development of the transformed epithelial phenotype.
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Anderson, Megan, and Aime A. Levesque. "Abstract 5644: Activation of p53 in prolonged prometaphase." Cancer Research 82, no. 12_Supplement (2022): 5644. http://dx.doi.org/10.1158/1538-7445.am2022-5644.
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Abstract The tumor suppressor p53 plays a critical role in protecting cells from aberrant divisions in the face of DNA damage, and while its role in DNA damage checkpoints have been extensively studied, its role in the mitotic checkpoint is less well characterized. The mitotic checkpoint prevents completion of mitosis until chromosomes have made bipolar kinetochore attachments to microtubules. Previous studies have shown that cells that experience a prolonged prometaphase(&gt;1.5 h) following treatment with microtubule destabilizing drugs will arrest in G1 following eventual exit from mitosis. Given its role in DNA damage checkpoints, p53 appears to be a likely candidate for involvement in this arrest. The goal of the current study is to investigate the role of p53 in this G1 arrest following prolonged prometaphase. Three cells lines were utilized:MCF10A, MCF10A/αp53 which overexpressed shRNA to p53, and MCF10A/OD which overexpresses the p53 oligomerization domain. Cells were treated with 9 μM RO-3306 for 18 h to synchronize cells at the G2/M transition. The RO-3306 was then removed and cells were treated with a microtubule destabilizing drug, nocodazole, at a concentration of 0.08 μM for 1.5 or 2.5 h to cause prometaphase arrest. The cells were then washed to remove nocodazole, and the cells were harvested at various time points and analyzed via flow cytometry and Western blot. Flow cytometry analysis in in all three cell lines showed an accumulation of cells in G1 at 12 hours in for both treatment times (1.5 and 2.5 h), with subsequent increases in sub-G1 populations by 24 hours. Western blot analysis revealed activation of p53 by phosphorylation of ser20 and activation of p21 in the MCF10A and MCF10A/OD, but not in the MCF10A/αp53 for both treatment times. Taken together, these results suggest that activation of p53 alone is not sufficient to elicit the G1 arrest, as the MCF10A/αp53 cells arrested in G1 despite the absence of p53 protein. Citation Format: Megan Anderson, Aime A. Levesque. Activation of p53 in prolonged prometaphase [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5644.
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Yoshimura, Fayth K., Tao Wang, Fei Yu, Hyeong-Reh C. Kim, and Jerrold R. Turner. "Mink Cell Focus-Forming Murine Leukemia Virus Infection Induces Apoptosis of Thymic Lymphocytes." Journal of Virology 74, no. 17 (2000): 8119–26. http://dx.doi.org/10.1128/jvi.74.17.8119-8126.2000.
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ABSTRACT In a previous study we identified the subpopulations of thymus cells that were infected by the lymphomagenic MCF13 murine leukemia virus (MLV) (F. K. Yoshimura, T. Wang, and M. Cankovic, J. Virol. 73:4890–4898, 1999) and observed an effect on thymus size by virus infection. In this report we describe our results which demonstrate that MCF13 MLV infection of thymuses reduced the number of T lymphocytes in this organ. Histological examination showed diffuse lymphocyte depletion, which was most striking in the CD4+CD8+ lymphocyte-enriched cortical zone. Consistent with this, flow cytometric analysis showed that the lymphocytes which were depleted were predominantly the immature CD3−CD4+ CD8+ and CD3+ CD4+CD8+ cells. A comparison of the percentages of live, apoptotic, and dead cells of the gp70+ and gp70− thymic lymphocytes suggested that this effect on thymus cellularity is a result of virus infection. Studies of the survival of thymic T lymphocytes in culture showed that cells from MCF13 MLV-inoculated mice underwent greater apoptosis and death than cells from control animals. Assays for apoptosis included 7-amino-actinomycin D staining, DNA fragmentation, and cleavage of caspase-3 and poly(ADP-ribose) polymerase proenzymes. Our results suggest that apoptosis of thymic lymphocytes by virus infection is an important step in the early stages of MCF13 MLV tumorigenesis.
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Gilles, Christine, Myriam Polette, Christelle Coraux та ін. "Contribution of MT1-MMP and of human laminin-5 γ2 chain degradation to mammary epithelial cell migration". Journal of Cell Science 114, № 16 (2001): 2967–76. http://dx.doi.org/10.1242/jcs.114.16.2967.
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Membrane-type matrix metalloproteinase 1 (MT1-MMP) is a membrane-anchored matrix metalloproteinase (MMP) that is frequently associated with processes involving tissue remodelling and cell migration. We have examined MT1-MMP expression and subcellular distribution as a function of MCF10A mammary epithelial cell migration using an in vitro outgrowth migration assay. Stronger expression of MT1-MMP was observed at the mRNA and at the protein level in cells at the periphery of the outgrowth. As shown by videomicroscopy,these cells were involved in an orientated cell migration, in contrast to stationary cells distant from the periphery. Furthermore, MT1-MMP was mainly distributed in lamellipodia of migratory cells, as well as at their basal surface in contact with the substrate. Laminin-5 (Ln-5), a recently described substrate for MT1-MMP, was deposited preferentially in the matrix by migratory cells. Fragments of the γ2 subunit of Ln-5 were also identified in migratory cultures of MCF10A cells, attesting to its proteolytic degradation. These fragments corresponded in size to those we observed after incubation of purified human Ln-5 with the recombinant catalytic domain of human MT1-MMP. We also show that anti-Ln5 blocking antibodies, MMP inhibitors (BB94 and TIMP-2)and MT1-MMP antisense oligonucleotides significantly decreased MCF10A cell migration. Taken together, these observations demonstrate that MT1-MMP is spatially and temporally regulated during MCF10A cell migration, and suggest that MT1-MMP-mediated pericellular proteolysis of Ln-5 γ2 chain could contribute to this process.
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Cheng, Qingsu, Mina Khoshdeli, Bradley S. Ferguson, Kosar Jabbari, Chongzhi Zang, and Bahram Parvin. "YY1 is a cis-regulator in the organoid models of high mammographic density." Bioinformatics 36, no. 6 (2019): 1663–67. http://dx.doi.org/10.1093/bioinformatics/btz812.
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Abstract Motivation Our previous study has shown that ERBB2 is overexpressed in the organoid model of MCF10A when the stiffness of the microenvironment is increased to that of high mammographic density (MD). We now aim to identify key transcription factors (TFs) and functional enhancers that regulate processes associated with increased stiffness of the microenvironment in the organoid models of premalignant human mammary cell lines. Results 3D colony organizations and the cis-regulatory networks of two human mammary epithelial cell lines (184A1 and MCF10A) are investigated as a function of the increased stiffness of the microenvironment within the range of MD. The 3D colonies are imaged using confocal microscopy, and the morphometries of colony organizations and heterogeneity are quantified as a function of the stiffness of the microenvironment using BioSig3D. In a surrogate assay, colony organizations are profiled by transcriptomics. Transcriptome data are enriched by correlative analysis with the computed morphometric indices. Next, a subset of enriched data are processed against publicly available ChIP-Seq data using Model-based Analysis of Regulation of Gene Expression to predict regulatory transcription factors. This integrative analysis of morphometric and transcriptomic data predicted YY1 as one of the cis-regulators in both cell lines as a result of the increased stiffness of the microenvironment. Subsequent experiments validated that YY1 is expressed at protein and mRNA levels for MCF10A and 184A1, respectively. Also, there is a causal relationship between activation of YY1 and ERBB2 when YY1 is overexpressed at the protein level in MCF10A. Supplementary information Supplementary data are available at Bioinformatics online.
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Kaan, Dilek. "Expression and biochemical significance of Piwil2 in stem cell lines." Postępy Higieny i Medycyny Doświadczalnej 76, no. 1 (2022): 97–103. http://dx.doi.org/10.2478/ahem-2022-0009.
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Abstract Introduction P-element induced wimpy testis-like 2 (Piwil2) is in the Piwi gene family. Piwil2 has important roles in the self-renewal mechanism of stem cell induction and progression of numerous types of human malignancies such as lung, breast, colon, prostate, and cervical cancers. Glutathione S-transferase (GST) acts as detoxification in cancer metabolism. This study aimed to investigate the effects of the stem cell protein Piwil2 on MCF10A and MCF-7 at the GST activity levels. Materials/Methods MCF-7/Piwil2 and MCF10A/Piwil2, transfected with a plasmid carrying the Piwil2 gene, and non-transfected MCF-7 and MCF10A were cultured in a complete DMEM/F12 medium. GST A1 and P1 activity was determined in these cell lines using as substrates CDNB, EA respectively. Results According to experimental results, GST P1 activity decreased in the MCF-7/Piwil2 cells as compared with the non-transfected MCF-7 cells, however, MCF-7/Piwil2 cells demonstrated increases in GST A1 (total GST) activity. The statistically significant differences were found for the comparison of non-transfected MCF-7 and MCF-7/Piwil2 (p<0,0001), for GST enzyme activities by using CDNB and EA as substrates. These results were the same for the MCF10A cell line. Discussion It is shown for the first time that transfection studies may affect GST activity at the cellular mechanism level. The study contributes to determining the effect of transfection on GST isoenzymes and also how the Piwil2 gene may affect GST activity in the stem cell line.
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Park, Woo Hyun. "Overexpression of the Fanconi Anemia A Gene in Hela and MCF10A Cells." Korean Journal of Hematology 41, no. 1 (2006): 1. http://dx.doi.org/10.5045/kjh.2006.41.1.1.
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Junkins, Katherine, Angel Perez Martinez, Margaret Rodgers, Shelley A. Phelan, and Min Xu. "Abstract 6512: Label-free autofluorescence imaging reveals different metabolic responses to adverse growth conditions between normal and breast cancer cells lines." Cancer Research 83, no. 7_Supplement (2023): 6512. http://dx.doi.org/10.1158/1538-7445.am2023-6512.
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Abstract Cancer cells are metabolically distinct from normal cells, and can adapt differently to adverse growth conditions. We sought to compare normal MCF10A and Ras-transformed MCF10AT cells under various conditions (regular growth media, doxorubicin-treatment, and glucose-deprived media) by imaging endogenous cellular FAD and NADH fluorescence and comparing it to proliferation rates (measured by MTS assay) and chromatin structure (measured by Hoescht staining). Under normal growth conditions, MCF10AT cells displayed similar levels of FAD and higher levels of mitochondrial NADH than MCF10A cells. Elevated NADH in MCF10AT cells seems to reflect the preference for anaerobic glycolysis rather than oxidative phosphorylation (the so-called Warburg effect). Our MTS data showed that doxorubicin treatment did not significantly affect MCF10AT cell proliferation but led to a modest reduction in cell proliferation in the normal cell line. Likewise, in MCF10AT cells, doxorubicin treatment led to an increase in FAD and a decrease in NADH. Conversely, in MCF10A cells, doxorubicin led to a decrease in FAD levels and an increase in mitochondrial NADH. These results suggest that the cancer line may be switching to oxidative phosphorylation to resist doxorubicin-induced toxicity, while the normal cells reduce oxidative phosphorylation with growth inhibition. In contrast to the doxorubicin response, glucose deprivation produced significant growth inhibition and cell death in MCF10AT cells while having a modest growth-inhibitory effect on the normal line. Interestingly, low glucose decreased FAD and mitochondrial NADH in MCF10AT cells, suggesting that both aerobic and anaerobic respiration was reduced and consistent with the marked reduction in observed cell viability. In contrast, MCF10A cells showed a slight decrease in FAD and an increase in NADH, suggesting a decrease in oxidative phosphorylation consistent with the modest growth inhibition observed. Finally, MCF10ATs displayed higher levels of Hoechst staining than MCF10A cells, consistent with a higher proportion of condensed chromatin observed in cancer cells. In addition, we found a larger increase in Hoechst staining in MCF10AT cells in response to both doxorubicin and low glucose, as compared to MCF10A cells. This data suggests that the chromatin state of the two lines is also differentially affected by these treatments. In conclusion, label-free measurement of autofluorescence of cellular FAD and mitochondrial NADH reveals the different metabolic and chromatin responses to adverse growth conditions between cancer and non-cancer cells. These differences are associated with the different proliferation rates and may have important clinical implications for cancer risk stratification. Citation Format: Katherine Junkins, Angel Perez Martinez, Margaret Rodgers, Shelley A. Phelan, Min Xu. Label-free autofluorescence imaging reveals different metabolic responses to adverse growth conditions between normal and breast cancer cells lines. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6512.
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Bougen-Zhukov, Nicola, Yasmin Nouri, Tanis Godwin, et al. "Allosteric AKT Inhibitors Target Synthetic Lethal Vulnerabilities in E-Cadherin-Deficient Cells." Cancers 11, no. 9 (2019): 1359. http://dx.doi.org/10.3390/cancers11091359.
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The CDH1 gene, encoding the cell adhesion protein E-cadherin, is one of the most frequently mutated genes in gastric cancer and inactivating germline CDH1 mutations are responsible for hereditary diffuse gastric cancer syndrome (HDGC). Using cell viability assays, we identified that breast (MCF10A) and gastric (NCI-N87) cells lacking CDH1 expression are more sensitive to allosteric AKT inhibitors than their CDH1-expressing isogenic counterparts. Apoptosis priming and total apoptosis assays in the isogenic MCF10A cells confirmed the enhanced sensitivity of E-cadherin-null cells to the AKT inhibitors. In addition, two of these inhibitors, ARQ-092 and MK2206, preferentially targeted mouse-derived gastric Cdh1−/− organoids for growth arrest. AKT protein expression and activation (as measured by phosphorylation of serine 473) were differentially regulated in E-cadherin-null MCF10A and NCI-N87 cells, with downregulation in the normal breast cells, but upregulation in the gastric cancer cells. Bioinformatic analysis of the TCGA STAD dataset revealed that AKT3, but not AKT1 or AKT2, is upregulated in the majority of E-cadherin-deficient gastric cancers. In conclusion, allosteric AKT inhibitors represent a promising class of drugs for chemoprevention and chemotherapy of cancers with E-cadherin loss.
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Olea-Flores, Monserrat, Miriam Zuñiga-Eulogio, Arvey Tacuba-Saavedra, et al. "Leptin Promotes Expression of EMT-Related Transcription Factors and Invasion in a Src and FAK-Dependent Pathway in MCF10A Mammary Epithelial Cells." Cells 8, no. 10 (2019): 1133. http://dx.doi.org/10.3390/cells8101133.
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Leptin is one of the main adipokines secreted in breast tissue. Leptin promotes epithelial–mesenchymal transition (EMT), cell migration and invasion in epithelial breast cells, leading to tumor progression. Although, the molecular mechanisms that underlie these events are not fully understood, the activation of different signaling pathways appears to be essential. In this sense, the effects of leptin on the activation of kinases like Src and FAK, which regulate signaling pathways that activate the EMT program, are not completely described. Therefore, we investigated the involvement of these kinases using an in vitro model for leptin-induced EMT process in the non-tumorigenic MCF10A cell line. To this end, MCF10A cells were stimulated with leptin, and Src and FAK activation was assessed. Specific events occurring during EMT were also evaluated in the presence or absence of the kinases’ chemical inhibitors PP2 and PF-573228. For instance, we tested the expression and subcellular localization of the EMT-related transcription factors Twist and β-catenin, by western blot and immunofluorescence. We also evaluated the secretion and activation of matrix metalloproteases (MMP-2 and MMP-9) by gelatin zymography. Invasiveness properties of leptin-stimulated cells were determined by invadopodia formation assays, and by the Transwell chamber method. Our results showed that leptin promotes EMT through Src and FAK activation, which leads to the secretion and activation of MMP-2 and MMP-9, invadopodia formation and cell invasion in MCF10A cells. In conclusion, our data suggest that leptin promotes an increase in the expression levels of Twist and β-catenin, the secretion of MMP-2, MMP-9, the invadopodia formation and invasion in MCF10A cells in a Src and FAK-dependent manner.
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Lyakhovich, Alex, and Malathy P. V. Shekhar. "Supramolecular Complex Formation between Rad6 and Proteins of the p53 Pathway during DNA Damage-Induced Response." Molecular and Cellular Biology 23, no. 7 (2003): 2463–75. http://dx.doi.org/10.1128/mcb.23.7.2463-2475.2003.
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ABSTRACT The HR6A and -B genes, homologues of the yeast Rad6 gene, encode ubiquitin-conjugating enzymes that are required for postreplication repair of DNA and damage-induced mutagenesis. Using surface plasmon resonance, we show here that HR6 protein (referred as Rad6) physically interacts with p53. Analysis of proteins coimmunoprecipitated with Rad6 antibody from metabolically labeled normal MCF10A human breast epithelial cells not only confirmed Rad6-p53 interactions in vivo but also demonstrated for the first time that exposure of MCF10A cells to cisplatin or adriamycin (ADR) induces recruitment of p14ARF into Rad6-p53 complexes. Further analysis of ADR-induced p53 response showed that stable Rad6-p53-p14ARF complex formation is associated with a parallel increase and decrease in monoubiquitinated and polyubiquitinated p53, respectively, and arrest in G2/M phase of the cell cycle. Interestingly, the ADR-induced suppression of p53 polyubiquitination correlated with a corresponding decline in intact Hdm2 protein levels. Treatment of MCF10A cells with MG132, a 26S proteasome inhibitor, effectively stabilized monoubiquitinated p53 and rescued ADR-induced downregulation of Hdm2. These data suggest that ADR-induced degradation of Hdm2 occurs via the ubiquitin-proteasome pathway. Rad6 is present in both the cytoplasmic and nuclear compartments of normal MCF10A cells, although in response to DNA damage it is predominantly found in the nucleus colocalizing with ubiquitinated p53, whereas Hdm2 is undetectable. Consistent with in vivo data, results from in vitro ubiquitination assays show that Rad6 mediates addition of one (mono-) to two (multimono-) ubiquitin molecules on p53 and that inclusion of Mdm2 is essential for its polyubiquitination. The data presented in the present study suggest that Rad6-p53-p14ARF complex formation and p53 ubiquitin modification are important damage-induced responses that perhaps determine the fidelity of DNA postreplication repair.
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Botos, Jeannine, Roger Smith, and Deborah T. Kochevar. "Retinoblastoma Function is a Better Indicator of Cellular Phenotype in Cultured Breast Adenocarcinoma Cells than Retinoblastoma Expression." Experimental Biology and Medicine 227, no. 5 (2002): 354–62. http://dx.doi.org/10.1177/153537020222700508.
Full textAbstract:
Loss of or lowered retinoblastoma (Rb) expression has been included as a prognostic indicator in breast cancer. Low or no Rb expression is seen most commonly in high-grade breast adenocarcinomas, suggesting that a relationship may exist between loss of Rb and a less differentiated state, high proliferation rate, and high metastatic potential. In this study, we compared Rb function in two established breast adenocarcinoma cell lines, MCF-7 and MDA-MB-231, and in an established immortalized mammary epithelial cell line, MCF10A. Cells were synchronized in G0/G1 and were released for several durations, at which time total Rb protein, mRNA, and Rb/E2F/DNA complex formation were evaluated. Rb protein was significantly higher in the tumor cells than in MCF10A cells. However, Rb function was high for a longer duration in MCF10A cells as compared with MCF-7 and MDA-MB-231 cells. Our data support the general conclusion that Rb function, but not necessarily Rb protein, is lower in highly malignant breast adenocarcinoma cells as compared with lower grade tumor cells. These results emphasize the relevance of assessing Rb function over Rb protein. This is particularly important if Rb is to be used as a prognostic indicator for breast adenocarcinoma.
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Risha, Yousef, Vanessa Susevski, Nico Hüttmann, Suttinee Poolsup, Zoran Minic, and Maxim V. Berezovski. "Breast Cancer-Derived Microvesicles Are the Source of Functional Metabolic Enzymes as Potential Targets for Cancer Therapy." Biomedicines 9, no. 2 (2021): 107. http://dx.doi.org/10.3390/biomedicines9020107.
Full textAbstract:
Membrane-derived extracellular vesicles, referred to as microvesicles (MVs), have been proposed to participate in several cancer diseases. In this study, MV fractions were isolated by differential ultracentrifugation from a metastatic breast cancer (BC) cell line MDA-MB-231 and a non-cancerous breast cell line MCF10A, then analyzed by nano-liquid chromatography coupled to tandem mass spectrometry. A total of 1519 MV proteins were identified from both cell lines. The data obtained were compared to previously analyzed proteins from small extracellular vesicles (sEVs), revealing 1272 proteins present in both MVs and sEVs derived from the MDA-MB-231 cell line. Among the 89 proteins unique to MDA-MB-231 MVs, three enzymes: ornithine aminotransferase (OAT), transaldolase (TALDO1) and bleomycin hydrolase (BLMH) were previously proposed as cancer therapy targets. These proteins were enzymatically validated in cells, sEVs, and MVs derived from both cell lines. The specific activity of OAT and TALDO1 was significantly higher in MDA-MB-231-derived MVs than in MCF10A MVs. BLMH was highly expressed in MDA-MB-231-derived MVs, compared to MCF10A MVs. This study shows that MVs carry functional metabolic enzymes and provides a framework for future studies of their biological role in BC and potential in therapeutic applications.