Non-tumorigenic Mammary Epithelial Cell Line Research Articles

PI 3-Kinase and the Histone Methyl-Transferase KMT2D Collaborate to Induce Arp2/3-Dependent Migration of Mammary Epithelial Cells

Breast cancer develops upon sequential acquisition of driver mutations in mammary epithelial cells; however, how these mutations collaborate to transform normal cells remains unclear in most cases. We aimed to reconstitute this process in a particular case. To this end, we combined the activated form of the PI 3-kinase harboring the H1047R mutation with the inactivation of the histone lysine methyl-transferase KMT2D in the non-tumorigenic human mammary epithelial cell line MCF10A. We found that PI 3-kinase activation promoted cell-cycle progression, especially when growth signals were limiting, as well as cell migration, both in a collective monolayer and as single cells. Furthermore, we showed that KMT2D inactivation had relatively little influence on these processes, except for single-cell migration, which KMT2D inactivation promoted in synergy with PI 3-kinase activation. The combination of these two genetic alterations induced expression of the ARPC5L gene that encodes a subunit of the Arp2/3 complex. ARPC5L depletion fully abolished the enhanced migration persistence exhibited by double-mutant cells. Our reconstitution approach in MCF10A has thus revealed both the cell function and the single-cell migration, and the underlying Arp2/3-dependent mechanism, which are synergistically regulated when KMT2D inactivation is combined with the activation of the PI 3-kinase.

An Increase in HSF1 Expression Directs Human Mammary Epithelial Cells toward a Mesenchymal Phenotype.

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.

In vitro cytotoxic effect of stigmasterol derivatives against breast cancer cells

BackgroundStigmasterol is an unsaturated phytosterol that belong to the class of tetracyclic steroids abundant in Rhoicissus tridentata. Stigmasterol is an important constituent since it has shown impressive pharmacological effects such as anti-osteoarthritis, anticancer, anti-diabetic, anti-inflammatory, antiparasitic, immunomodulatory, antifungal, antioxidant, antibacterial, and neuroprotective activities. Furthermore, due to the presence of π system and hydroxyl group, stigmasterol is readily derivatized through substitution and addition reactions, allowing for the synthesis of a wide variety of stigmasterol derivatives.MethodsStigmasterol (1) isolated from Rhoicissus tridentata was used as starting material to yield eight bio-active derivatives (2–9) through acetylation, epoxidation, epoxide ring opening, oxidation, and dihydroxylation reactions. The structures of all the compounds were established using spectroscopic techniques, NMR, IR, MS, and melting points. The synthesized stigmasterol derivatives were screened for cytotoxicity against the hormone receptor-positive breast cancer (MCF-7), triple-negative breast cancer (HCC70), and non-tumorigenic mammary epithelial (MCF-12 A) cell lines using the resazurin assay.ResultsEight stigmasterol derivatives were successfully synthesized namely; Stigmasterol acetate (2), Stigmasta-5,22-dien-3,7-dione (3), 5,6-Epoxystigmast-22-en-3β-ol (4), 5,6-Epoxystigmasta-3β,22,23-triol (5), Stigmastane-3β,5,6,22,23-pentol (6), Stigmasta-5-en-3,7-dion-22,23-diol (7), Stigmasta-3,7-dion-5,6,22,23-ol (8) and Stigmast-5-ene-3β,22,23-triol (9). This is the first report of Stigmasta-5-en-3,7-dion-22,23-diol (7) and Stigmasta-3,7-dion-5,6,22,23-ol (8). The synthesized stigmasterol analogues showed improved cytotoxic activity overall compared to the stigmasterol (1), which was not toxic to the three cell lines tested (EC50 ˃ 250 µM). In particular, 5,6-Epoxystigmast-22-en-3β-ol (4) and stigmast-5-ene-3β,22,23-triol (9) displayed improved cytotoxicity and selectivity against MCF-7 breast cancer cells (EC50 values of 21.92 and 22.94 µM, respectively), while stigmastane-3β,5,6,22,23-pentol (6) showed improved cytotoxic activity against the HCC70 cell line (EC50: 16.82 µM).ConclusionNatural products from Rhoicissus tridentata and their derivatives exhibit a wide range of pharmacological activities, including anticancer activity. The results obtained from this study indicate that molecular modification of stigmasterol functional groups can generate structural analogues with improved anticancer activity. Stigmasterol derivatives have potential as candidates for novel anticancer drugs.

Cytotoxic Effect of Phenylethanoid Glycosides Isolated from Plantago lanceolata L.

The aim of the study is to investigate whether the bioactive compounds isolated from P. lanceolata inflorescences, namely, phenylethanoid glucosides, acteoside, plantamajoside, and a flavonoid, isorhamnetin-3-O-rutinoside-4'-O-glucoside, possessed cytotoxic activity against the selected cancer cell lines. The potential antitumor effects of two phenylethanoid glycosides and one flavonoid were evaluated via MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay on seven human carcinoma cell lines (MCF-7, MDA-MB-231, Caco-2, HepG2, OVCAR-3, U138-MG, U251-MG) and one nontumorigenic mammary epithelial cell line (MCF-12A). For the first time, acteoside was studied in ovarian cancer cell line OVCAR-3, and plantamajoside in all cell lines except breast adenocarcinoma MDA-MB-281 and hepatocarcinoma HepG2. The phenylethanoid glycosides showed stronger cytotoxic activity than that of the glycoside flavonoid. Acteoside and plantamajoside, at concentrations of 200 and 300 μM, respectively, had a highly toxic effect on the selected two cancer cell lines of breast adenocarcinoma MDA-MB-231 and MCF-7, ovarian cancer cell line OVCAR-3, glioblastoma cell line U138-MG, and hepatocarcinoma cell line HepG2. Both glycosides were significantly less cytotoxic towards nontumorigenic cell line MCF-12A; the effect appeared at a concentration of 400 μM. For the first time, the activity of acteoside and plantamajoside was compared in one parallel investigation. The results are discussed against a broad background of existing knowledge on biological effects, their mechanisms, and structure-activity relationships. Phenylethanoids may be potential compounds with cytotoxic activity against the selected cancer types.

13-amino derivatives of dehydrocostus lactone display greatly enhanced selective toxicity against breast cancer cells and improved binding energies to protein kinases in silico.

The biological activities of dehydrocostus lactone and its analogues are suggested to be mediated by the lactone ring and α,β-methylene-γ-lactone. However, few studies exist on the structure-activity relationship of 13-amino derivatives of dehydrocostus latone. In this study new 13-amino derivatives of dehydrocostus lactone DHLC (1–4) were synthesized through Michael addition reactions, and were screened against three different breast cancer cell lines, namely hormone receptor positive breast cancer (MCF-7), triple-negative breast cancer (HCC70), and non-tumorigenic mammary epithelial (MCF-12A) cell lines. Dehydrocostus lactone (DHLC) exhibited IC50 values of 1.11 (selectivity index (SI) = 0.06), 24.70 (SI = 0.01) and 0.07 μM against HCC70, MCF-7 and MCF-12A cells, respectively. All the amino derivatives, except DHLC-3 displayed low micromolar IC50 values (ranging from 0.07–4.24 μM) against both breast cancer cell lines, with reduced toxicity towards MCF-12A non-tumorigenic mammary epithelial cells (SI values ranging from 6.00–126.86). DHLC-1 and DHLC-2 demonstrated the greatest selectivity for the MCF-7 cells (with SI of 121 and 126.86 respectively) over the MCF-12A cells. This reveals that, overall, the derivatives display greatly improved selectivity for breast cancer over non-tumorigenic mammary epithelial cells, with between 100-fold and 12 000-fold higher SI values. The improved docking scores were recorded for all the 13-amino dehydrocostus lactone derivatives for the enzymes analyzed. Compounds DHLC-4, and DHLC-3 recorded higher docking scores of -7.33 and -5.97 Kca/mol respectively, compared to the parent structure, dehydrocostus lactone (-5.34 Kca/mol) for protein kinase (PKC) theta (1XJD) and -6.22 and -5.88 Kca/mol, respectively for protein kinase iota (1RZR). The compounds further showed promising predicted adsorption, distribution, metabolisms and excretion (ADME) properties. Predicting the ADME properties of these derivatives is of importance in evaluating their drug-likeness, which could in turn be developed into potential drug candidates.

Photodynamic therapy with the dual-mode association of IR780 to PEG-PLA nanocapsules and the effects on human breast cancer cells.

IR780 is a near-infrared fluorescent dye, which can be applied as a photosensitizer in photodynamic (PDT) and photothermal (PTT) therapies and as a biodistribution tracer in imaging techniques. We investigated the growth and migration inhibition and mechanism of death of breast tumor cells, MCF-7 and MDA-MB-231, exposed to polymeric nanocapsules (NC) comprising IR780 covalently linked to the biodegradable polymer PLA (IR-PLA) and IR780 physically encapsulated (IR780-NC) in vitro. Both types of NC had mean diameters around 120nm and zeta potentials around -40mV. IR-PLA-NC was less cytotoxic than IR780 NC to a non-tumorigenic mammary epithelial cell line, MCF-10A, which is an important aspect of selectivity. Free-IR780 was more cytotoxic than IR-PLA-NC for MCF-7 and MDA-MB-231 cells after illumination with a 808nm laser. IR-PLA NC was effective to inhibit colony formation (50%) and migration (30-40%) for both cancer cell lines. MDA-MB-231 cells were less sensitive to all IR780 formulations compared to MCF-7 cells. Cell uptake was higher with IR-PLA-NC than with IR780-NC and free-IR780 in both cancer cell lines (p<0.05). NC uptake was higher in MCF-7 than in MDA-MB-231 cells. IR-PLA-NC induced a higher percentage of apoptosis upon illumination in MDA-MB-231 than in MCF-7 cells. The necrosis mechanism of death predominated in treatments with free-IR780 and with encapsulated IR780 NC, suggestive of damages at the plasma membrane. IR780 conjugated with PLA increased the apoptotic pathway and demonstrated potential as a multifunctional theranostic agent for breast cancer treatment with increased cellular uptake, photodynamic activity and more reliable tracking in cell-image studies.

Biological activity of Haematoxylum brasiletto in MCF7 and MDA-MB-231 breast cancer cell lines

Haematoxylum brasiletto (Fabaceae) is a wild plant from Guerrero, used in traditional medicine. However, there is little scientific information about anti-migratory and antioxidant activities. The aim of this study was to evaluate the anti-migratory and anti-proliferative activities of the ethanolic extract of heartwood of H. brasiletto (HB) and their fractions of hexane (HBA) dichloromethane (HBB) and ethyl acetate (HBC) against different breast cancer cell lines. Additionally, we analyzed their antioxidant activity. The antiproliferative activity of ethanolic extract and their fractions were evaluated against the non-tumorigenic mammary epithelial cell line MCF10A and the cancer cell lines MCF7 and MDA-MB-231 by MTT assay, using concentrations of 3.1 to 25 μg/mL for 48 h. The anti-migratory activity was evaluated by the scratch wound-healing assay. Phenolic content (TPC) was quantified by the Folin-Ciocalteu assay, and flavonoid content (TFC) was investigated using the AlCl3 assay. Antioxidant activity was evaluated through the DPPH, trolox equivalent antioxidant capacity (TEAC), and ferric reducing antioxidant power (FRAP) methods. Our results showed that HB (258.6mg GAE/g) presented the higher amount of TPC. For TFC, we found that HBC (53.0 mg QE/g) presented the higher content. Antioxidant evaluation results evidenced that HBC [DPPH IC50: 57 μg/mL; TEAC: 61.4 μM TE/g; FRAP: 2678.2 μM Fe(II)/g] exhibited the most remarkable capacity to stabilize free radicals and reduce metals. On the other hand, HBB was the one who presented the most anti-proliferative and anti-migratory activities on MCF7. In addition, extract and fractions was less cytotoxic against the non-cancerous MCF1710 A cells, which may be associated with their selectivity against cancer cells. To our knowledge, this is the first study that reports the activity of the H. brasiletto plant. Besides, H. brasiletto extracts and their fractions showed an interesting anti-proliferative, anti-migratory activity, and antioxidant potential, which could be considered as a natural source of antioxidants and anti-cancer compounds.

In vitro cytotoxic effects of chemical constituents of Euphorbia grandicornis Blanc against breast cancer cells

Euphorbia grandicornis Blanc is widely utilized in traditional medicine for a variety of ailments including body pains associated with skin irritations, inflammation, and snake or scorpion bites. Compounds from E. grandicornis were characterized using spectroscopic techniques, NMR, IR, MS, and melting points and alongside the extracts were evaluated for in vitro anticancer activity against several cancer cell lines. The root extract afforded known, β-glutinol (1), β-amyrin (2), 24-methylenetirucalla-8-en-3β-ol (3), tirucalla-8,25-diene-3β,24R-diol (4), stigmasterol (5), sitosterol (6), and hexyl (E)-3-(4‑hydroxy-3-methoxyphenyl)-2-propenoate (7) based on their NMR spectroscopic data for the first report in E. grandicornis. The extracts and isolated compounds were evaluated for anticancer activities against hormone receptor-positive breast cancer (MCF-7), triple-negative breast cancer (HCC70), and non-tumorigenic mammary epithelial (MCF-12A) cell lines. The CH2Cl2 extract exhibited potent, cytotoxicity against MCF-7, HCC70, and MCF-12A cells. The aerial extract exhibited IC50 values of 1.03, 0.301, and 1.68 µg/mL, and root extract displayed IC50 values of 0.83, 0.83 and 3.98 µg/mL against MCF-7, HCC70, and MCF-12A cells, respectively. The root extract thus showed selectivity for the cancer cell lines over the non-cancerous control cell line (SI = 4.80). Hexyl (E)-3-(4‑hydroxy-3-methoxyphenyl)-2-propenoate (7) showed significant activity with IC50 values of 23.41, 29.45 and 27.01 µM against MCF-7, HCC70 and MCF-12A cells, respectively, suggesting non-specific cytotoxicity.

DNAJC25 as a tumor suppressor candidate gene in breast cancer

DNAJC25 gene is a member of the HSP40 (DNAJ) family, and it was suggested as a tumor suppressor gene in hepatocellular carcinoma. The aim of this study was to analyze the expression, genetic/epigenetic alterations, and prognostic value of the DNAJC25 gene in breast cancer. DNAJC25 transcript levels are upregulated in BT-20 and ZR-75-1 cell lines and downregulated in MDA-MB-231 cell line compared to the non-tumorigenic mammary epithelial cell line (MCF 10A) (P&lt; 0.05). According to UALCAN in-silico tool, clinical breast cancer samples show significantly reduced levels of DNAJC25 mRNA levels relative to the normal samples (P=1.47e-02). The Kaplan–Meier plotter tool shows that high DNAJC25 expression is favorable for post-progression survival in breast cancer (P=0.0035). Point mutations or copy number variations of DNAJC25 are uncommon in clinical breast cancer samples. Combined bisulfite restriction analysis (COBRA) results showed that DNAJC25 promoter is not methylated in breast cell lines. Promoter hypomethylation was also observed in normal and tumor clinical samples (Beta-value &lt; 0.25). In conclusion, DNAJC25 is suggested as a tumor suppressor candidate having limited biomarker potential in breast cancer. Functional studies are essential to reveal its role in breast carcinogenesis.

Ultrastructural Analysis of Inflammatory Breast Cancer Cell Clusters in an Ex Vivo Environment Mechanically Mimicking the Lymph Vascular System.

Background:Inflammatory breast cancer (IBC) is a rare form of breast cancer with a poor prognosis. IBC is characterized by florid lymphovascular tumor emboli in the skin and the parenchyma of the breast. We hypothesized that the formation of these emboli/clusters plays a pivotal role in IBC metastasis and its rapid progression, and that their structure and function may be a key to identifying molecular biological differences between IBC and non IBC.Methods:Mechanical methods were used to mimic the lymph fluid viscosity by adding 2.25% of PEG8000 to the media. Clusters were obtained for IBC tumor cell lines (SUM149 and IBC-3), non IBC tumor cell lines (MDA-MB-231, MDA-MB-468, and MCF7), and a non-tumorigenic human mammary epithelial cell line (MCF10A). Clusters were analyzed by light microscopy, and then prepared for and observed by transmission electron microscopy (TEM).Results:Significant differences were seen between IBC and non IBC clusters. The TEM analysis revealed that IBC cells harbored numerous microvilli and microvesicles, both on the free outer surface and inside the cluster. Microvilli from IBC cell clusters were noted at higher density and were longer than those of non IBC cell clusters.Conclusions:IBC tumor cell clusters exhibited distinct ultrastructural features characterized by the presence of long, crowded microvilli and numerous microvesicles. These microvilli may play an important role in the biology and aggressiveness of IBC.

RNA-seq reveals novel cancer-selective and disease subtype-independent mechanistic targets of withaferin A in human breast cancer cells.

Withaferin A (WA) exhibits cancer chemopreventive efficacy in preclinical models representative of two different subtypes of breast cancer. However, the mechanism(s) underlying breast cancer chemoprevention by WA is not fully elucidated. We performed RNA-seq analyses using a non-tumorigenic mammary epithelial cell line (MCF-10A) and human breast cancer cells (BCC) belonging to the luminal-type (MCF-7), HER2-enriched (SK-BR-3), and basal-like subtype (MDA-MB-231) to identify novel cancer-selective mechanistic targets of WA. The WA-regulated transcriptome was strikingly different between MCF-10A versus BCC. The Kyoto Encyclopedia of Genes and Genomes pathway analysis revealed downregulation of genes associated with cellular senescence in WA-treated BCC. Consequently, the number of senescence-associated β-galactosidase positive cells was decreased significantly in WA-treated BCC but not in the MCF-10A cells. WA treatment caused upregulation of senescence marker p21 more robustly in BCC than in MCF-10A. Breast cancer prevention by WA in rats was also associated with upregulation of p21 protein expression. The Reactome pathway analyses indicated upregulation of genes associated with cellular response to stress/external stimuli in WA-treated BCC but not in MCF-10A. Two proteins represented in these pathways (HSPA6 and NRF2) were further investigated. While HSPA6 was dispensable for WA-mediated apoptosis and autophagy or inhibition of cell migration, the NRF2 knockout cells were more resistant to apoptosis resulting from WA treatment than control cells. Finally, expression of some glycolysis-related proteins was decreased by WA treatment both in vitro and in vivo. In summary, this study provides novel insights into cancer-selective pathways affected by WA that may contribute to its chemopreventive efficacy in breast cancer.

Assessment of replication of bovine herpesvirus type 4 in human glioblastoma and breast cancer cells as a potential oncolytic virus.

Oncolytic viruses have been extensively used in cancer treatment due to their tropism, selective replication only in tumor cells, and possible synergic interaction with other therapeutics. Different researchers have demonstrated that bovine herpesvirus 4 (BoHV-4), a member of the gammaherpesviridae family, has oncolytic potential in some human-origin cancer cell lines like glioma through the selective replication strategy. Using four apoptosis detection methods, namely MTT, LDH, TUNEL, and Annexin V assays, we evaluated the apoptotic effect of BoHV-4 Movar33/63 reference strain along with a recombinant BoHV-4 expressing EGFP in U87 MG cells (human glioblastoma cell line), MDA MB-231 (human breast cancer cell line), and MCF10a (non-tumorigenic human mammary epithelial cell line). Our findings indicate that this virus can replicate and induce apoptosis in these cell lines and hinder in vitro proliferation in a dose-dependent manner. In conclusion, BoHV-4 has in vitro potential as a novel oncolytic virus in human cancer therapy. However, its replication potential in the MCF10a cells as a non-tumorigenic human mammary epithelial cell line is a concern in using this virus in cancer therapy, at least against human mammary tumors. Further studies must therefore be conducted to examine the specific apoptotic pathways induced by this virus to move on to further experiments.

Downregulation of DNAJC10 (ERDJ5) is associated with poor survival in breast cancer.

DNAJC10 (ERDJ5), a member of HSP40 family, was considered as an anti-oncogenic gene in neuroblastoma, prostate and colon cancers. But, the role and importance of DNAJC10 gene in breast cancer is currently unknown. In this study, in vitro/in vivo expression, biomarker potential and genetic/epigenetic alterations of DNAJC10 were analyzed in breast cancer. Real-time qRT-PCR and immunohistochemistry methods were used to determine the expression level of DNAJC10 gene in breast cancer cell lines and clinical samples. The Kaplan-Meier plotter was used to evaluate the survival prognostic value of DNAJC10 mRNA expression in breast cancer patients. Mutation screening software and methylation-specific PCR were used to screen genetic alterations and methylation status of DNAJC10 promoter regions, respectively. DNAJC10 mRNA expression was significantly reduced in 3 out of 4 breast cancer cell lines compared to the nontumorigenic mammary epithelial cell line (MCF 10A). DNAJC10 protein expression was significantly less frequent in invasive ductal carcinoma samples (n = 121) compared with adjacent normal breast tissues (n = 32) (p < 0.0001). Downregulation of DNAJC10 mRNA was associated with poor overall survival (OS) (n = 626) (p = 0.0096) and relapse-free survival (n = 1764) (p = 5.3e-12). According to the COSMIC and cBioPortal databases, point mutations and copy number variations of DNAJC10 were very rare in breast cancer samples. Besides, no genetic alterations on the experimentally validated promoter regions were found in breast cell lines. CpG island located in the promoter regions of DNAJC10 gene was found to be frequently hypomethylated in breast cell lines. In the light of previous knowledge regarding the role of DNAJC10 in carcinogenesis, findings of this study suggest that DNAJC10 is a potential diagnostic/prognostic biomarker and tumor suppressor candidate for breast cancer. Epigenetic factors other than promoter methylation could contribute to the downregulation of DNAJC10 expression.

The Leptin induced Hic-5 expression and actin puncta formation by the FAK/Src-dependent pathway in MCF10A mammary epithelial cells

ResumenIntroducción. La leptina es una hormona secretada por los adipocitos que se ha relacionado con el proceso de la transición de epitelio a mesénquima (Epithelial- Mesenchymal Transition, EMT). Promueve la migración e invasión de las células del epitelio mamario mediante la activación de las cinasas FAK y Src, un complejo regulador de vías de señalización que favorecen la expresión de las proteínas relacionadas con la formación de estructuras proteolíticas implicadas en la invasión y progresión del cáncer. Recientemente, se ha descrito que la sobreexpresión y activación de la proteína Hic-5 durante el mencionado proceso de transición, favorece la formación de los puntos de actina (indicativa de la formación y funcionalidad de los invadopodios), lo cual promueve la degradación local de los componentes de la matriz extracelular y la metástasis del cáncer.Objetivos. Evaluar el papel de las cinasas FAK y Src sobre la expresión y localización subcelular de Hic-5 y la formación de puntos de actina inducida por la leptina en la línea celular MCF10A de epitelio mamario no tumoral.Materiales y métodos. Se utilizaron los inhibidores específicos de la FAK (PF-573228) y la Src (PP2) para evaluar el papel de ambas cinasas en los niveles de expresión y localización subcelular de la proteína Hic-5 mediante Western blot e inmunofluorescencia, así como la formación de puntos de actina mediante la tinción con faloidina-TRITC en células MCF10A estimuladas con leptina.Resultados. La leptina indujo el incremento en la expresión de Hic-5 y la formación de puntos de actina. El tratamiento previo con los inhibidores de las cinasas FAK (PF-573228) y Src (PP2), promovió la disminución en la expresión de Hic-5 y de los puntos de actina en la línea celular MCF10A de epitelio mamario no tumoral.Conclusión. La leptina indujo la expresión y la localización perinuclear de Hic-5 y la formación de puntos de actina mediante un mecanismo dependiente de la actividad de las cinasas FAK y Src en las células MCF10A.

Tumor suppressor role of cytoplasmic polyadenylation element binding protein 2 (CPEB2) in human mammary epithelial cells

BackgroundOver-expression of cyclooxygenase (COX)-2 promotes breast cancer progression by multiple mechanisms, including induction of stem-like cells (SLC). Combined gene expression and microRNA microarray analyses of empty vector vs COX-2- transfected COX-2 low MCF7 breast cancer cell line identified two COX-2-upregulated microRNAs, miR-526b and miR-655, both found to be oncogenic and SLC-promoting. Cytoplasmic Polyadenylation Element-Binding Protein 2 (CPEB2) was the single common target of both microRNAs, the functions of which remain controversial. CPEB2 has multiple isoforms (A-F), and paradoxically, a high B/A ratio was reported to impart anoikis-resistance and metastatic phenotype in triple- negative breast cancer cells. We tested whether CPEB2 is a tumor suppressor in mammary epithelial cells.MethodsWe knocked-out CPEB2 in the non-tumorigenic mammary epithelial cell line MCF10A by CRISPR/Cas9-double nickase approach, and knocked-down CPEB2 with siRNAs in the poorly malignant MCF7 cell line, both lines being high CPEB2-expressing. The resultant phenotypes for oncogenity were tested in vitro for both lines and in vivo for CPEB2KO cells. Finally, CPEB2 expression was compared between human breast cancer and non-tumor breast tissues.ResultsCPEB2 (isoform A) expression was inversely correlated with COX-2 or the above microRNAs in COX-2-divergent breast cancer cell lines. CPEB2KO MCF10A cells exhibited oncogenic properties including increased proliferation, migration, invasion, EMT (decreased E-Cadherin, increased Vimentin, N-Cadherin, SNAI1, and ZEB1) and SLC phenotype (increased tumorsphere formation and SLC marker-expression). Tumor-suppressor p53 protein was shown to be a novel translationally-regulated target of CPEB2, validated with polysome profiling. CPEB2KO, but not wild-type cells produced lung colonies upon intravenous injection and subcutaneous tumors and spontaneous lung metastases upon implantation at mammary sites in NOD/SCID/IL2Rϒ-null mice, identified with HLA immunostaining. Similarly, siRNA-mediated CPEB2 knockdown in MCF7 cells promoted oncogenic properties in vitro. Human breast cancer tissues (n = 105) revealed a lower mRNA expression for CPEB2 isoform A and also a lower A/B isoform ratio than in non-tumour breast tissues (n = 20), suggesting that CPEB2A accounts for the tumor-suppressor functions of CPEB2.ConclusionsCPEB2, presumably the isoform A, plays a key role in suppressing tumorigenesis in mammary epithelial cells by repressing EMT, migration, invasion, proliferation and SLC phenotype, via multiple targets, including a newly-identified translational target p53.

Anti-tumor properties of methoxylated analogues of resveratrol in malignant MCF-7 but not in non-tumorigenic MCF-10A mammary epithelial cell lines

Resveratrol is a plant-derived polyphenol that is known for its anti-inflammatory and anti-tumorigenic properties in in vitro and in vivo models. Recent studies show that some resveratrol analogues might be more potent anti-tumor agents, which may partly be attributed to their ability to activate the aryl hydrocarbon receptor (AHR). Here, the anti-tumorigenic properties of resveratrol and structural analogues oxyresveratrol, pinostilbene, pterostilbene and tetramethoxystilbene (TMS) were studied in vitro, using in the malignant human MCF-7 breast cancer cell line and non-tumorigenic breast epithelial cell line MCF-10A.Cell viability and migration assays showed that methoxylated analogues of resveratrol are more potent anti-tumorigenic compounds than resveratrol and its hydroxylated analogue oxyresveratrol, with 2,3’,4,5’-tetramethoxy-trans-stilbene (TMS) being the most potent compound. TMS decreased MCF-7 tumor cell viability with 50% at 3.6 μM and inhibited migration with 37.5 ± 14.8% at 3 μM. In addition, TMS activated the AHR more potently (EC50 in a reporter gene assay 2.0 μM) and induced AHR-mediated induction of cytochrome P450 1A1 (CYP1A1) activity (EC50 value of 0.7 μM) more than resveratrol and the other analogues tested. Cell cycle analysis showed that TMS induced a shift in cell cycle status from the G1 to the G2/M phase causing a cell cycle arrest in the MCF-7 cells, while no effect of TMS was observed in the non-tumorigenic MCF-10A mammary epithelial cell line. Gene expression analysis showed that 3 μM TMS increased gene expression of CYP1A1 (289-fold), CYP1B1 (5-fold) and Nqo1 (2-fold), and decreased gene expression of IL-8 (3-fold) in MCF-7 cells. In MCF-10A cells, 10 μM TMS also increased gene expression of CYP1A1 (5-fold) and CYP1B1 (2-fold), but decreased gene expression of Nqo1 (1.4-fold) in contrast to MCF-7 cells.TMS displays more potent anti-tumorigenic properties and activates the AHR more effectively than resveratrol. In addition, this is the first study to show that TMS, but not resveratrol, selectively inhibits the cell cycle of breast tumor cells and not the non-tumorigenic cells. Our study provides more insight in the anti-tumor properties of the methoxylated analogues of resveratrol in breast cells in vitro.

Abstract 4464: CPEB-2 is a tumor-suppressor gene in human breast cancer

Abstract We found that expression of cyclooxygenase (COX)-2 promotes breast cancer progression by multiple mechanisms, including stimulation of cancer cell proliferation, migration, invasiveness, epithelial-to-mesenchymal transition (EMT), angiogenesis, lymphangiogenesis and induction of stem-like cells (SLC). Through combined gene expression and microRNA microarray analyses of COX-2-transfected MCF-7 cells, we identified two oncogenic miRNAs, miR-526b and miR-655, up-regulated by COX-2. They collectively target 13 tumour suppressor-like genes down-regulated by COX-2, of which Cytoplasmic Polyadenylation Element-Binding Protein -2 (CPEB-2) was the single common target, whose function is unclear in breast cancer. CPEB-2 is known to downregulate mesenchymal transcription factor TWIST1 and translation of HIF1α. CPEB-2 has multiple isoforms (A-F), of which a high B: A isoform ratio was reported to confer anoikis-resistance, a metastatic phenotype, in breast cancer cells. We found that CPEB-2 expression was inversely correlated with COX-2 or the microRNAs miR-526b and miR-655 in breast cancer cell lines; it was very high in a non-tumorigenic mammary epithelial cell line MCF10A.To define its functional roles we knocked out CPEB-2 in MCF10A cells by CRISPR/Cas9-double nickase approach. CPEB-2-KO cells exhibited oncogenic phenotypes in vitro: increased proliferation, migration, invasion, EMT phenotype (decreased E-Cadherin, increased Vimentin, N-cadherin, SNAI1, and ZEB1) and SLC properties (increased spheroid formation and SLC-linked markers). P53 protein was found to be a novel translationally- regulated target of CPEB-2. CPEB-2KO cells, but not wild-type cells, produced lung colonies upon intravenous injection and orthotopic tumors (identified by HLA staining) upon implantation at mammary sites in NOD/SCID/IL2Rϒ-null mice. In human breast cancer tissues, compared to non-tumour breast tissues, isoform A/E of CPEB2 revealed a lower expression in HER2+ breast cancer samples, suggesting that CPEB-2A/E are the tumour-suppressor isoforms. These findings demonstrate that CPEB-2 is a tumour suppressor gene in breast cancer. (Supported by the OICR and the NSERC) Citation Format: Peeyush K. Lala, Joshua Tordjman, Mehdi Amiri, Asma Hasan, David Hess, Mousumi Majumder. CPEB-2 is a tumor-suppressor gene in human breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4464.

Abstract 3269A: 2-Hydroxyflavanone: A novel estrogen receptor alpha downregulator with potent antitumor effect in breast cancer

Abstract Breast cancer (BC) remains one of the major causes of cancer deaths in women. 2Hydroxyflavanone (2HF), a constituent of citrus fruits, inhibits growth of human breast cancer cell lines in culture. The present study was undertaken to determine in vivo efficacy of 2HF against MDAMB231 human breast cancer xenografts. The 2HF administration retarded growth of MDAMB231 cells subcutaneously implanted in female nude mice without causing weight loss or any other side effects. The 2HF mediated suppression of MDAMB231 xenograft growth correlated with reduced cell proliferation as revealed by immunohistochemical analysis for Ki67 expression. Analysis of the vasculature in the tumors from 2HF treated mice indicated smaller vessel area compared with control tumors based on immunohistochemistry for angiogenesis marker CD31. The 2HF mediated inhibition of angiogenesis in vivo correlated with downregulation of vascular endothelial growth factor (VEGF) level in the tumor. Consistent with these results, 2HF treatment suppressed cell cycle regulators levels in cultured MDAMB231 cells. Moreover, the 2HF treated MDAMB231 cells exhibited reduced capacity for migration compared with vehicle-treated control cells. In addition, 2HF administration elicits apoptotic response as judged by annexin V and PI staining and TUNEL assay. 2HF treatment reduced the levels of ER alpha in ER positive MCF7 breast cancer cells. In conclusion, the present study demonstrates in vivo anticancer efficacy of 2HF against MDAMB231 xenografts in association with reduced cell proliferation and suppression of neovascularization. Our results indicate that 2HF is a promising anticancer agent for breast cancer due to its ability to collectively inhibit major regulators of breast cancer including RLIP76, ER alpha, and HER2. Interestingly, a spontaneously immortalized non-tumorigenic normal human mammary epithelial cell line (MCF10A) is significantly more resistant to 2HF mediated apoptosis compared with breast cancer cells, demonstrating selectivity of 2HF towards malignant breast cells. These preclinical observations merit clinical investigation to determine efficacy of 2HF against human breast cancers. (This work was supported in part by the Department of Defense grant W81XWH 16 1 0641. Funding from the Beckman Research Institute of City of Hope is also acknowledged). Citation Format: Sharad S. Singhal, Shireen Chikara, Lokesh Nagaprashantha, Jyotsana Singhal, David Horne, Sanjay Awasthi. 2-Hydroxyflavanone: A novel estrogen receptor alpha downregulator with potent antitumor effect in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3269A.

Characterization of immortalized human mammary epithelial cell line HMEC 2.6.

Primary human mammary epithelial cells have a limited life span which makes it difficult to study them in vitro for most purposes. To overcome this problem, we have developed a cell line that was immortalized using defined genetic elements, and we have characterized this immortalized non-tumorigenic human mammary epithelial cell line to establish it as a potential model system. human mammary epithelial cells were obtained from a healthy individual undergoing reduction mammoplasty at SIU School of Medicine. The cells were transduced with CDK4R24C followed by transduction with human telomerase reverse transcriptase. Post all manipulation, the cells displayed a normal cell cycle phase distribution and were near diploid in nature, which was confirmed by flow cytometry and karyotyping. In vitro studies showed that the cells were anchorage dependent and were non-invasive in nature. The cell line expressed basal epithelial markers such as cytokeratin 7, CD10, and p63 and was negative for the expression of estrogen receptor and progesterone receptor. Upon G-band karyotyping, the cell line displayed the presence of a few cytogenic abnormalities, including trisomy 20 and trisomy 7, which are also commonly present in other immortalized mammary cell lines. Furthermore, the benign nature of these cells was confirmed by multiple in vitro and in vivo experiments. Therefore, we think that this cell line could serve as a good model to understand the molecular mechanisms involved in the development and progression of breast cancer and to also assess the effect of novel therapeutics on human mammary epithelial cells.

Cytokine profile of breast cell lines after different radiation doses

Purpose: Ionizing radiation (IR) treatment activates inflammatory processes causing the release of a great amount of molecules able to affect the cell survival. The aim of this study was to analyze the cytokine signature of conditioned medium produced by non-tumorigenic mammary epithelial cell line MCF10A, as well as MCF7 and MDA-MB-231 breast cancer cell lines, after single high doses of IR in order to understand their role in high radiation response.Materials and methods: We performed a cytokine profile of irradiated conditioned media of MCF10A, MCF7 and MDA-MB-231 cell lines treated with 9 or 23 Gy, by Luminex and ELISA analyses.Results: Overall, our results show that both 9 Gy and 23 Gy of IR induce the release within the first 72 h of cytokines and growth factors potentially able to influence the tumor outcome, with a dose-independent and cell-line dependent signature. Moreover, our results show that the cell-senescence phenomenon does not correlate with the amount of ‘senescence-associated secretory phenotype’ (SASP) molecules released in media. Thus, additional mechanisms are probably involved in this process.Conclusions: These data open the possibility to evaluate cytokine profile as useful marker in modulating the personalized radiotherapy in breast cancer care.