Wild-type MCF-7 Cells Research Articles

Simultaneous delivery of anti-miR21 with doxorubicin prodrug by mimetic lipoprotein nanoparticles for synergistic effect against drug resistance in cancer cells.

The development of drug resistance in cancer cells is one of the major obstacles to achieving effective chemotherapy. We hypothesized that the combination of a doxorubicin (Dox) prodrug and microRNA (miR)21 inhibitor might show synergistic antitumor effects on drug-resistant breast cancer cells. In this study, we aimed to develop new high-density lipoprotein-mimicking nanoparticles (HMNs) for coencapsulation and codelivery of this potential combination. Dox was coupled with a nuclear localization signal (NLS) peptide to construct a prodrug (NLS-Dox), thereby electrostatically condensing miR21 inhibitor (anti-miR21) to form cationic complexes. The HMNs were formulated by shielding these complexes with anionic lipids and Apo AI proteins. We have characterized that the coloaded HMNs had uniformly dispersed distribution, favorable negatively charged surface, and high coencapsulation efficiency. The HMN formulation effectively codelivered NLS-Dox and anti-miR21 into Dox-resistant breast cancer MCF7/ADR cells and wild-type MCF7 cells via a high-density-lipoprotein receptor-mediated pathway, which facilitated the escape of Pgp drug efflux. The coloaded HMNs consisting of NLS-Dox/anti-miR21 demonstrated greater cytotoxicity with enhanced intracellular accumulation in resistant MCF7/ADR cells compared with free Dox solution. The reversal of drug resistance by coloaded HMNs might be attributed to the suppression of miR21 expression and the related antiapoptosis network. Furthermore, the codelivery of anti-miR21 and NLS-Dox by HMNs showed synergistic antiproliferative effects in MCF7/ADR-bearing nude mice, and was more effective in tumor inhibition than other drug formulations. These data suggested that codelivery of anti-miR21 and chemotherapeutic agents by HMNs might be a promising strategy for antitumor therapy, and could restore the drug sensitivity of cancer cells, alter intracellular drug distribution, and ultimately enhance chemotherapeutic effects.

Methylation of Notch3 modulates chemoresistance via P-glycoprotein

The global gene expression and DNA methylation of genes in adriamycin-resistant human breast cancer cells (MCF-7/ADM cells) are similar to those in paclitaxel-resistant MCF-7 cells (MCF-7/PTX) and are significantly different from those in wild-type MCF-7 cells. DNA methylation is associated with chemoresistance in breast cancer and changes the characteristics of chemoresistant and chemosensitive cells. Here, we showed that the tumor-suppressor gene Notch3 was inactivated due to epigenetic silencing DNA hypermethylation in MCF-7/ADM cells. In addition, the drug efflux pump P-glycoprotein was negatively regulated by Notch3 and highly expressed in MCF-7/ADM cells. Taken together, our findings demonstrated that hypermethylation of Notch3 causes activation of P-glycoprotein in adriamycin-resistant cells.

11a-N-Tosyl-5-deoxi-pterocarpan, LQB-223, a novel compound with potent antineoplastic activity toward breast cancer cells with different phenotypes.

Multidrug resistance is the major obstacle for successful treatment of breast cancer, prompting the investigation of novel anticancer compounds. In this study, we tested whether LQB-223, an 11a-N-Tosyl-5-deoxi-pterocarpan newly synthesized compound, could be effective toward breast cancer cells. Human breast cell lines MCF-7, MDA-MB-231, HB4a and MCF-7 Dox(R) were used as models for this study. Cell culture, MTT and clonogenic assay, flow cytometry and Western blotting were performed. The LQB-223 decreased cell viability, inhibited colony formation and induced an expressive G2/M arrest in breast cancer cells. There was an induction in p53 and p21(Cip1) protein levels following treatment of wild-type p53 MCF-7 cells, which was not observed in the mutant p53 MDA-MB-231 cell line, providing evidence that the compound might act to modulate the cell cycle regardless of p53 status. In addition, LQB-223 resulted in decreased procaspase levels and increased annexin V staining, suggesting that the apoptotic cascade is also triggered. Importantly, LQB-223 treatment was shown to be less cytotoxic to non-neoplastic breast cells than docetaxel and doxorubicin. Strikingly, exposure of doxorubicin-resistant MCF-7-Dox(R) cells to LQB-223 resulted in suppression of cell viability and proliferation in levels comparable to MCF-7. Of note, MCF-7-Dox(R) cells have an elevated expression of the P-glycoprotein efflux pump when compared to MCF-7. Together, these results show that LQB-223 mediates cytotoxic effects in sensitive and resistant breast cancer cells, while presenting low toxicity to non-neoplastic cells. The new compound might represent a potential strategy to induce toxicity in breast cancer cells, especially chemoresistant cells.

Abstract LB-113: Antihormone-induced expression of BCL3 in estrogen receptor-positive breast cancer drives resistant cell growth

Abstract Introduction: The development of resistance to antihormones in the treatment of oestrogen receptor-positive (ER+) breast cancer is a major clinical problem. There is emerging evidence that antihormones can induce expression of genes/proteins that allow a cohort of cells to evade the growth-inhibitory impact of these therapies during initial drug response and ultimately aid acquisition of resistant cell growth. In the present study we have examined antihormone-induced expression of the pro-survival/pro-proliferative gene BCL3 and its potential role in the development of resistance. Methods: ER+ MCF-7, T47D, BT474 and MDAMB361 cells were exposed to antihormone treatment and effects on BCL3 expression were examined. BCL3 expression was also examined in cell models of resistance to tamoxifen (TAMR) and oestrogen deprivation (XR). BCL3 siRNA knockdown studies were performed on wild-type MCF-7, TAMR and XR cell models and impact on p50 expression and EGFR/HER2/MAPK signalling was assessed by Western blotting. Cell survival and growth was also assessed using FACS analysis and cell counting. Cellular localisation and association of BCL3 and p50 in antihormone-responsive and -resistant MCF-7 cells was also determined by immunofluorescence and immunoprecipitation. Results: BCL3 mRNA and protein expression was markedly increased in response to all antihormonal treatments in our panel of four ER+ breast cancer cell lines and elevated BCL3 levels were also observed in the TAMR and XR cell models. Surprisingly, transient BCL3 knockdown by siRNA in TAMR cells had no effect on cell survival but reduced cell proliferation. This growth inhibitory effect of BCL3 knockdown was also observed to a lesser degree in XR but not in wild-type MCF-7 cells. BCL3 knockdown was without effect on previously identified growth regulatory signalling proteins in our TAMR cells but was associated with loss of p50 expression, suggesting the identification of a novel NF-κB-mediated resistant growth regulatory pathway. Immunofluorescence revealed nuclear expression of BCL3 and p50 during resistance, however, during response BCL3 was located in the nucleus and p50 in the cytoplasm. In support of these findings, immunoprecipitation studies identified an association between BCL3 and p50 during resistance which was absent during antihormone response. Conclusion: These results demonstrate that BCL3 is induced by antihormones during response and induction is maintained through to resistance. Interestingly, BCL3 appears to have a growth regulatory role in antihormone-resistant but not wild-type MCF-7 cells possibly due to its association with p50 in resistance, thus potentially activating transcription of target genes involved in cell proliferation. These findings suggest that targeting BCL3 may provide a novel therapeutic strategy to treat resistant disease; however its role in response remains to be elucidated. Citation Format: Jessica Davis, Janice Knowlden, Chris Pepper, Julia Gee, Iain Hutcheson. Antihormone-induced expression of BCL3 in estrogen receptor-positive breast cancer drives resistant cell growth. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-113.

Abstract 2921: The retinoblastoma protein regulates hypoxia-inducible factor-1α-mediated transcriptional programs, tumor cell invasiveness, tumor growth and metastasis in human breast cancer cells

Abstract The retinoblastoma protein (Rb) is capable of attenuating the hypoxic response in tumor cells. This process is mediated by the hypoxia inducible factor 1α/2α (HIF1α/2α) and its dimerization partner the aryl hydrocarbon receptor nuclear translocator (ARNT/ HIF1β). Rb modulates HIF activity by virtue of its association with the thyroid hormone receptor/retinoblastoma interacting protein 230 (TRIP230), an essential cofactor of the HIF1α/ARNT transcriptional complex. We used short hairpin RNA (shRNA) technology and microarray analysis to interrogate the Rb-negative and wild-type MCF7 cell transcriptomes and generated lists of genes that were either up- or downregulated in response to both loss of Rb and hypoxia. We found that loss of Rb enhances the expression of hypoxia-regulated genes involved in invasion and epithelial-to-mesenchymal transition and significantly decreases the expression of genes involved in cell anchoring and differentiation in MCF7 and MDA-MB-231 breast cancer cells. Genes were validated using both qRT-PCR and immuno-blot analysis. Additionally, gene ontology analysis revealed that AKT and ERK1/2 are downstream effectors of hypoxic gene programs that are sensitive to loss of Rb. Furthermore, these factors regulate the acquisition of a more invasive phenotype in breast cancer cells. Finally, we found that Rb knockdown in combination with pre-treatment of cells with hypoxia increased growth of tumor foci in the lungs after i.v. injection and increased the development of spontaneous metastases from orthotopically implanted breast tumor cells in female NOD-SCID mice. Primary tumors lacking Rb demonstrated enriched protein levels of genes identified in our arrays when compared to negative control tumors. These results show that Rb is a negative modulator of hypoxia-regulated genetic programs by virtue of its direct effects on the HIF-complex. Understanding the HIF complex and the molecular mechanisms controlling the progression from benign tumors to metastasized and lethal forms will allow us to develop more specific breast cancer therapies. Citation Format: Mandeep K. Takhar, Mark P. Labrecque, Kevin J. Tam, Anne Haegert, Robert H. Bell, Manuel Altamirano-Dimas, Colin C. Collins, Gratien G. Prefontaine, Michael E. Cox, Kevin L. Bennewith, Timothy V. Beischlag. The retinoblastoma protein regulates hypoxia-inducible factor-1α-mediated transcriptional programs, tumor cell invasiveness, tumor growth and metastasis in human breast cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2921.

Global regulation of alternative RNA splicing by the SR-rich protein RBM39

BackgroundRBM39 is a serine/arginine-rich RNA-binding protein that is highly homologous to the splicing factor U2AF65. However, the role of RBM39 in alternative splicing is poorly understood. MethodsIn this study, RBM39-mediated global alternative splicing was investigated using RNA-Seq and genome-wide RBM39-RNA interactions were mapped via cross-linking and immunoprecipitation coupled with deep sequencing (CLIP-Seq) in wild-type and RBM39-knockdown MCF-7 cells. ResultsRBM39 was involved in the up- or down-regulation of the transcript levels of various genes. Hundreds of alternative splicing events regulated by endogenous RBM39 were identified. The majority of these events were cassette exons. Genes containing RBM39-regulated alternative exons were found to be linked to G2/M transition, cellular response to DNA damage, adherens junctions and endocytosis. CLIP-Seq analysis showed that the binding site of RBM39 was mainly in proximity to 5′ and 3′ splicing sites. Considerable RBM39 binding to mRNAs encoding proteins involved in translation was observed. Of particular importance, ~20% of the alternative splicing events that were significantly regulated by RBM39 were similarly regulated by U2AF65. ConclusionsRBM39 is extensively involved in alternative splicing of RNA and helps regulate transcript levels. RBM39 may modulate alternative splicing similarly to U2AF65 by either directly binding to RNA or recruiting other splicing factors, such as U2AF65. General significanceThe current study offers a genome-wide view of RBM39's regulatory function in alternative splicing. RBM39 may play important roles in multiple cellular processes by regulating both alternative splicing of RNA molecules and transcript levels.

EFFECT OF 5-AZACYTIDINE ON miRNA EXPRESSION IN HUMAN BREAST CANCER CELLS WITH DIFFERENT SENSITIVITY TO CYTOSTATICS

To analyze expression of miRNA in human breast cancer cells, sensitive and resistant to cisplatin and doxorubicin, and to explore possible modification of drug sensitivity via treatment of cells with 5-azacytidine (5-aza), a demethylating agent. The study was performed on wild-type MCF-7 cell line (MCF-7/S) and its two sublines MCF-7/Dox and MCF-7/DDP resistant to doxorubicin and cisplatin, respectively. Cells were treated with 5-aza, cisplatin, doxorubicin and their combinations. Relative expression levels of miRNA-221, -200b, -320a, -10b, -34a, -122 and -29b were examined, using qRT-PCR. The MTT assay was used to monitor cell viability. We compared miRNA expression profiles in MCF-7/S and drug resistant MCF-7/Dox and MCF-7/DDP cells. Changes of miRNA-221, -200b, -320a, -10b, -34a, -122 and -29b were observed in both resistant cell lines. The most significant differences were found for miRNA-200b (decreased in 50.0 ± 2.6 and 63.0 ± 3.1 times for MCF-7/Dox and MCF-7/DDP cells, respectively) and for oncogenic miRNA-221 levels (increase in 62.0 ± 5.7 times for MCF-7/Dox and 83.8 ± 7.2 times for MCF-7/DDP cells). 5-aza treatment caused an increase of miRNA-10b, -122, -200b levels in MCF-7/S cells, miRNA-34a, -10b, -122, -200b and -320a levels in MCF-7/Dox cells and miRNA-34a, -10b, -200b and -320a levels in MCF-7/DDP cells. Pretreatment of all studied lines with 5-aza resulted in the increase of their sensitivity to studied cytostatics. In particular, the IC50 of doxorubicin decreased by 2-, 4- and 3-fold for cell lines MCF-7/S, MCF-7/Dox and MCF-7/DDP cells, respectively, and IC50 of cisplatin in studied cultures decreased by 3-, 2- and 1.5-fold, respectively. It was shown that use of 5-aza can modify sensitivity of breast cancer cells to cytotoxic drugs not only by it's demetylation effect, but also by changes in expression of miRNAs, involved in cell proliferation, migration and drug resistance development.

Abstract P4-08-06: Modulation of hypoxia-inducible factors and the HIF transcriptional response to hypoxia by ERBB2 overexpression in the MCF7 breast cancer cell line

Abstract Objective: To explore the role of HIF2α in growth factor receptor-driven HIF modulation and investigate the relationship between growth factor- and hypoxia-driven HIF activation. HIF-mediated transcriptional activity is known to drive genes involved in various processes which are associated with cancer pathology such as glycolysis, angiogenesis and metastasis. Therefore, understanding the implications of hypoxia-independent HIF regulation for both HIF1α and HIF2α, may give new insight into the mechanisms by which HIF drives cancer pathology in vivo and a greater understanding of when HIF inhibitory agents may be effective therapies. Methods: We used an ERBB2 overexpressing MCF7 cell line (MCF7-HER2) to investigate the effect of ERBB2 on the HIF-axis. Western blotting was used to assess protein level in these cell lines. HIF protein expression was compared with and without ERBB stimulation by ERBB3 ligand neuregulin 1β. Illumina BeadChip analysis was used to compare mRNA levels between these cell lines in normoxia (20% oxygen), acute hypoxia (0.5% oxygen for 24 hours) and chronic hypoxia (0.5% oxygen for 10 weeks). Differentially expressed genes were identified using rank products analysis with a cut-off P-value of 0.01. This allowed an in-depth comparison of hypoxia responses at the level of transcription between the cell lines to ascertain the effect of ERBB2 overexpression on hypoxia driven transcriptional changes. Results: Immunoblotting shows that HIF1α protein level is comparable between MCF7 and MCF7-HER2 cell lines, and is inducible in normoxia by stimulation with neuregulin 1β. Conversely, HIF2α protein is unaffected, but is constitutively expressed in MCF7-HER2 only. This suggests that both HIF isoforms can be up-regulated in normoxia but by different mechanisms. Microarray data suggests that the constitutively higher HIF2α levels in the MCF7-HER2 cell line may be due, at least in part, to the increased transcription of the HIF2A gene which is higher in normoxia and in response to hypoxia when compared to wild-type MCF7. Overexpression of ERBB2 in MCF7-HER2 cells appears to prime cells for their response to hypoxia, as 14% (N= 591) of the genes which are induced in acute hypoxia are also expressed at significantly higher levels in normoxic MCF7-HER2 cells. However, only 1% are more highly expressed in wild-type MCF7 cells. For chronic hypoxic genes, 18% (N= 514) were more highly expressed in normoxic MCF7-HER2 cells and just 8% in wild-type MCF7 cells. These up-regulated genes include both HIF1 and HIF2 target genes which may have important consequences for glycolysis (ALDOC, PFKFB), tumour cell survival (E4BP4, STC2) and proliferation (FOS, KDM5B). Conclusions: We have demonstrated that both HIF1α and HIF2α can be regulated independently of hypoxia, however these appear to be controlled through distinct mechanisms. Whilst the implications of HIF1 in breast cancer pathology have been appreciated for some time, relatively little is known about the impact of HIF2. Here we show that ERBB2 overexpression can not only increase HIF2α protein levels in normoxia, but may also prime cells for hypoxia by allowing the constitutively higher expression of HIF1 and HIF2 target genes. Citation Format: Jarman EJ, Turnbull AK, Martinez-Perez C, Meehan J, Xintralopoulou C, Ward C, Langdon SP. Modulation of hypoxia-inducible factors and the HIF transcriptional response to hypoxia by ERBB2 overexpression in the MCF7 breast cancer cell line. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P4-08-06.

Acquision of Resistance to Doxorubicin By Breast Cancer Cells MCF7 Enhances Their Procoagulant Properties and Alters the Efficacy of Antithrombotic Agents to Inhibit Thrombin Generation

Introduction: A major problem associated with breast cancer chemotherapy is the subsequent development of resistance to chemotherapeutic agents (multidrug résistance-MDR). Hypercoagulability, increased risk of venous thromboebolism (VTE) and high rate of failure of the antithrombotic treatment with LMWHs have been observed in patients with disease resistant to the chemotherapy.Aim of the study: In the present study we investigated if acquisition of MDR by breast cancer cells MCF7 is associated with modification of their procoagulant potency and induces alteration of the efficacy of the antithrombotic agents. We evaluated the capacity of wild type MCF7 cells (MCF7/WT) and doxorubicin resistant cells (MCF7/DR) to trigger thrombin generation (TG) and to modify the antithrombotic activity of the LMWH enoxaparin, and the specific direct and indirect FXa inhibitors (apixaban and fondaparinux).Materials and Methods: Pre-treatment of MCF-7 cells for several weeks with increasing concentrations of doxorubicin, induced the acquisition of chemo-resistance phenotype documented by the expression the MDR1-Pgp. Tissue factor (TF) and MDR1-Pgp expression by MCF7/WT and MCF7/DR cells were assessed by flow cytometry and western blot assays. Reverse transcriptase - polymerase chain reaction (RT-PCR) for TF mRNA expression was also performed. Thrombin generation of normal platelet poor plasma (PPP) in the presence of MCF7 cells was assessed with the Calibrated Automate Thrombogram® assay (Diagnostica Stago). TG in the presence of MCF7 cells was also assessed in PPP spiked with clinically relevant concentrations of enoxaparin, apixaban or fondaparinux. The efficiency of the studied agents in the presence of MCF7 cells was compared to that in the control experiment where TG was triggered by PPP-Reagent® 5 pM TF. The antithrombotic agents were compared on the bases of the concentration which inhibited 50% TG (IC50).Results: The MCF7/DR cells showed higher expression of TF as compared to MCF7/WT. The TF expression by MCF7/DR was correlated with the expression of the MDR1/P-gp. The MCF7/DR cells significantly enhanced TG as compared to MCF7/WT cells. The three studied agents significantly inhibited TG at plasma concentrations usually achieved at doses for thromboprophylaxis. The presence resistant MCF7 cells did not significantly modify the antithrombotic potency of fondaparinux. The inhibitory effect of enoxaparin and apixaban on thrombin generation was partially reversed when TG was triggered by MCF7/DR as compared to that triggered by MCF7/WT or in the control experiment.Table 1Modification of the inhibitory potency of the antithrombotic agents by MCF7 cells on thrombin generation in function of the acquisition of MDR phenotype.IC50 for MRIPPP + TF/PPLPPP + MCF7/WTPPP+ MCF7/DRfondaparinux (anti-Xa IU/mL)0.21 ± 0.030.28 ± 0.060.30 ± 0.04enoxaparin (anti-Xa IU/mL)0.10 ± 0.020.12 ± 0.050.23 ± 0.08*apixaban (ng/ml)14 ± 0.919 ± 3.126 ± 1.4**p<0.05 versus MCF7/WT PPLprocoagulant phospholipids MRImean rate of the propagation phase of thrombin generationIC50: concentration that inhibits 50% the MRIConclusions: The acquisition of the chemo-resistance phenotype by the breast cancer cells MCF7 is associated with enhancement of their procoagulant properties. The procoagulant fingerprint of the chemo-resistant MCF7 cells is characterized by increased expression of TF which is correlated with enhanced thrombin generation. The chemo-resistant MCF7 cells reverse the antithrombotic effect of enoxaparin and apixaban but not that of fondaparinux. DisclosuresVan Dreden:Diagnostica Stago: Employment.

Altered regulation of PDK4 expression promotes antiestrogen resistance in human breast cancer cells.

Acquired or de novo resistance to the selective estrogen receptor modulators tamoxifen and fulvestrant (ICI) is a major barrier to successful treatment of breast cancer. Gene expression patterns in tamoxifen resistant (TamR-MCF-7) cells were compared to their parental cells (MCF-7L) to identify an aberrantly regulated metabolic pathway. TamR-MCF-7 cells are cross resistant to ICI and doxorubicin, and have increased mitochondrial DNA. A small subset of genes had altered expression in TamR-MCF-7 relative to MCF-7L cells. One of the genes, pyruvate dehydrogenase kinase-4 (PDK4), phosphorylates pyruvate dehydrogenase (PDH). PDK4 expression was elevated in TamR-MCF-7 cells; this result was also observed in a second model of acquired antiestrogen resistance. PDK4 expression is controlled in part by glucocorticoid response elements in the PDK4 gene promoter. In MCF-7L cells, PDK4 mRNA expression was insensitive to glucocorticoid receptor agonists, while dexamethasone dramatically increased PDK4 expression in TamR-MCF-7 cells. Using siRNA to knock down PDK4 expression increased TamR-MCF-7 sensitivity to ICI; in contrast adapting cells to growth in glucose depleted media did not affect ICI sensitivity. Despite TamR-MCF-7 cells high levels of PDK4 mRNA relative to MCF-7L, TamR-MCF-7 cells have increased PDH activity. Wild type MCF-7 cells are reported to be heterozygous for a G to A mutation that results in a substitution of threonine for alanine near PDK4′s catalytic site. We found loss of heterozygosity in TamR-MCF-7 cells; TamR-MCF-7 are homozygous for the wild type allele. These data support a role for altered regulation of PDH by PDK4 and altered substrate utilization in the development of drug resistance in human breast cancer cells.Electronic supplementary materialThe online version of this article (doi:10.1186/s40064-015-1444-2) contains supplementary material, which is available to authorized users.

Integration of Downstream Signals of Insulin-like Growth Factor-1 Receptor by Endoplasmic Reticulum Stress for Estrogen-Induced Growth or Apoptosis in Breast Cancer Cells.

Estrogen (E2) exerts a dual function on E2-deprived breast cancer cells, with both initial proliferation and subsequent induction of stress responses to cause apoptosis. However, the mechanism by which E2 integrally regulates cell growth or apoptosis-associated pathways remains to be elucidated. Here, E2 deprivation results in many alterations in stress-responsive pathways. For instance, E2-deprived breast cancer cells had higher basal levels of stress-activated protein kinase, c-Jun N-terminal kinase (JNK), compared with wild-type MCF-7 cells. E2 treatment further constitutively activated JNK after 24 hours. However, inhibition of JNK (SP600125) was unable to abolish E2- induced apoptosis, whereas SP600125 alone arrested cells at the G2 phase of the cell cycle and increased apoptosis. Further examination showed that inhibition of JNK increased gene expression of TNFα and did not effectively attenuate expression of apoptosis-related genes induced by E2. A notable finding was that E2 regulated both JNK and Akt as the downstream signals of insulin-like growth factor-1 receptor (IGFIR)/PI3K, but with distinctive modulation patterns: JNK was constitutively activated, whereas Akt and Akt-associated proteins, such as PTEN and mTOR, were selectively degraded. Endoplasmic reticulum-associated degradation (ERAD) was involved in the selective protein degradation. These findings highlight a novel IGFIR/PI3K/JNK axis that plays a proliferative role during the prelude to E2-induced apoptosis and that the endoplasmic reticulum is a key regulatory site to decide cell fate after E2 treatment. This study provides a new rationale for further exploration of E2-induced apoptosis to improve clinical benefit.

Upregulation of BTG1 enhances the radiation sensitivity of human breast cancer in vitro and in vivo.

X-ray-based radiotherapy is one of the most effective therapeutic strategies for breast cancer patients. However, radioresistance and side-effects continue to be the most challenging issues. B-cell translocation gene 1 (BTG1) is a member of the BTG/Tob family, which inhibits cancer growth and promotes apoptosis. We, therefore, hypothesized that BTG1 plays an important role in the radiosensitivity of breast cancer cells. In the present study, breast cancer cell lines that stably overexpressed BTG1 were used to investigate the effects of BTG1 on cell radiosensitivity in vitro. We found that overexpression of BTG1 enhanced the radiosensitivity both of p53-mutant breast cancer MDA-MB-231 cells and p53 wild-type breast cancer MCF-7 cells. We also found that overexpression of BTG1 along with irradiation induced cell cycle G2/M phase arrest, promoted the formation of reactive oxygen species (ROS), increased the rate of chromosomal aberrations and increased cell apoptosis. Further investigation indicated that BTG1 overexpression along with irradiation was involved in inhibition of the PI3K/Akt signaling pathway. Importantly, the finding that BTG1 promoted ionizing radiosensitivity of breast cancer cells in vitro was confirmed in an animal model. Taken together, our data suggest that BTG1 overexpression combined with radiation therapy increases the therapeutic efficacy of breast cancer treatment via regulation of the cell cycle and apoptosis-related signaling pathways.

Activation of peroxisome proliferator-activated receptor gamma is crucial for antitumoral effects of 6-iodolactone.

BackgroundMolecular iodine (I2) exhibits antiproliferative and apoptotic effects on in vivo and in vitro cancer models. These effects are thought to be mediated by an iodinated arachidonic acid derivative, 6-iodolactone (6IL), and one of the proposed mechanisms is that 6IL activates Peroxisome Proliferator-Activated Receptors type gamma (PPARG). These receptors have been implicated in the inhibition of carcinogenic processes, in addition to their classical role in maintaining lipid and glucose homeostasis. The aim of this study was to determine whether PPARG participates in the 6IL antiproliferative and apoptotic effects on the mammary cancer cell line MCF-7.MethodsThe 6IL/PPARG complex was inhibited by the PPARG antagonist GW9662, in both an endogenous and overexpressed (adenoviral vector infection) context, and stable PPARG-knockdown MCF-7 cells (RNA interference, confirmed with hydrolysis probes and Western blot), were used to corroborate the PPARG participation. 6IL effects on proliferation (measured by Trypan Blue exclusion) and apoptosis (phosphatidylserine identification by flow cytometer) were evaluated in conditions of chemical inhibition (GW9662) and silencing (RNA interference). A wound-healing assay was conducted on wild-type and stable PPARG-knockdown MCF-7 cells to evaluate the antimigrational effect of 6IL. Caspase-8 activity was evaluated to determine if the extrinsic pathway is involved in the effects of 6IL and I2 treatment.ResultsAntiproliferative and pro-apoptotic 6IL effects require the activation of PPARG. In addition, wound-healing assays show that 6IL is able to inhibit MCF-7 cell migration and that PPARG plays a role in this phenomenon. Finally, the data exclude the participation of the extrinsic apoptotic pathway in 6IL- and I2-induced apoptosis.ConclusionsThese results support the previously proposed mechanism, in which the I2 effects are mediated by 6IL, and they provide further support for the use of I2 as coadjuvant in breast cancer treatment.

PIWI-interacting RNA 021285 is involved in breast tumorigenesis possibly by remodeling the cancer epigenome.

Although PIWI-interacting RNAs (piRNAs) account for the largest class of the small non-coding RNA superfamily, virtually nothing is known of their function in human carcinogenesis. Once thought to be expressed solely in the germ line where they safeguard the genome against transposon-induced insertional mutations, piRNAs are now believed to play an active role in somatic gene regulation through sequence-specific histone modification and DNA methylation. In the current study, we investigate the role of piRNA-021285 (piR-021285) in the regulation of the breast cancer methylome. Genotypic screening of a panel of single-nucleotide polymorphism (SNP)-containing piRNAs revealed a significant association between SNP rs1326306 G>T in piR-021285 and increased likelihood for breast cancer in a Connecticut-based population (441 cases and 479 controls). Given nascent but compelling evidence of piRNA-mediated gene-specific methylation in the soma, a genome-wide methylation screen was then carried out using wild type (WT) and variant piR-021285 mimic-transfected MCF7 cells to explore whether the observed association could be attributed in part to piR-021285-induced methylation at cancer-relevant genes. We found significant methylation differences at a number of experimentally implicated breast cancer-related genes, including attenuated 5' untranslated region (UTR)/first exon methylation at the proinvasive ARHGAP11A gene in variant mimic-transfected cells. Follow-up functional analyses revealed both concurrent increased ARHGAP11A mRNA expression and enhanced invasiveness in variant versus WT piR-021285 mimic-transfected breast cancer cell lines. Taken together, our findings demonstrate the first evidence supporting a role of piRNAs, a novel group of non-coding RNA, in human tumorigenesis via a piRNA-mediated epigenetic mechanism, which warrants further confirmation and investigation.

Inhibition of SDF-1/CXCR4-induced epithelial-mesenchymal transition by kisspeptin-10.

Recently we have shown that breast cancer cell invasion was dramatically increased when co-cultured with MG63 cells. In addition we have generated mesenchymal transformed MCF-7 breast cancer cells (MCF-7-EMT), showing significantly increased invasion in contrast to wild type MCF-7 cells (MCF-7 WT). In this study we have analyzed whether stromal derived factor-1 (SDF-1) is responsible for MCF-7 and T-47-D breast cancer cell invasion and epithelial-mesenchymal-transition (EMT). In addition we have analyzed whether kisspeptin-10 (KP-10) treatment affects SDF-1-induced invasion and EMT. Invasion was quantified by assessment of MCF-7 and T-47-D breast cancer cell migration rate through an artificial basement membrane in a modified Boyden chamber during co-culture with MG63 cells or after treatment with SDF-1α, SDF-1β or the combination of both isoforms. Induction of EMT was verified by analysis of protein expression of epithelial marker E-cadherin (CDH1) and mesenchymal markers N-cadherin (CDH2) and Vimentin (VIM). The role of SDF-1 for invasion and induction of EMT in breast cancer cells was analyzed by blocking SDF-1 secretion during co-culture with MG63 cells. In addition effects of KP-10 treatment on SDF-1-induced invasion and EMT were analyzed. Breast cancer cell invasion was significantly increased when co-cultured with MG63 cells. During co-culture SDF-1 protein expression of MG63 cells was significantly induced. The increased breast cancer cell invasion could be blocked by anti-SDF-1 antibodies. Treatment of breast cancer cells in monoculture (without MG63) with SDF-1α, SDF-1β or the combination of both isoforms resulted in a significant escalation of breast cancer cell invasion and induction of EMT. Protein expression of mesenchymal markers CDH2 and VIM was clearly elevated, whereas protein expression of epithelial marker CDH1 was clearly decreased. The SDF-1-induced increase of cell invasion was significantly reduced after treatment with KP-10. In addition, induction of EMT was inhibited. Furthermore, protein expression of the binding site of SDF-1, CXC-motive-chemokine receptor 4 (CXCR-4), was reduced by KP-10. Treatment of MCF-7-EMT cells with KP-10 resulted in a significant drop of cell invasion and CXCR-4 protein expression. Our findings suggest that SDF-1 plays a major role in breast cancer invasion and EMT. SDF-1-induced invasion and EMT can be inhibited by KP-10 treatment by down-regulating CXCR-4 expression.

Thyroid Hormone Receptors Predict Prognosis in BRCA1 Associated Breast Cancer in Opposing Ways.

Since BRCA1 associated breast cancers are frequently classified as hormone receptor negative or even triple negative, the application of endocrine therapies is rather limited in these patients. Like hormone receptors that bind to estrogen or progesterone, thyroid hormone receptors (TRs) are members of the nuclear hormone receptor superfamily. TRs might be interesting biomarkers - especially in the absence of classical hormone receptors. The current study aimed to investigate whether TRs may be specifically expressed in BRCA1 associated cancer cases and whether they are of prognostic significance in these patients as compared to sporadic breast cancer cases. This study analyzed TRα and TRβ immunopositivity in BRCA1 associated (n = 38) and sporadic breast cancer (n = 86). Further, TRs were studied in MCF7 (BRCA1 wildtype) and HCC3153 (BRCA1 mutated) cells. TRβ positivity rate was significantly higher in BRCA1 associated as compared to sporadic breast cancers (p = 0.001). The latter observation remained to be significant when cases that had been matched for clinicopathological criteria were compared (p = 0.037). Regarding BRCA1 associated breast cancer cases TRβ positivity turned out to be a positive prognostic factor for five-year (p = 0.007) and overall survival (p = 0.026) while TRα positivity predicted reduced five-year survival (p = 0.030). Activation of TRβ resulted in down-modulation of CTNNB1 while TRα inhibition reduced cell viability in HCC3153. However, only BRCA1 wildtype MCF7 cells were capable of rapidly degrading TRα1 in response to T3 stimulation. Significantly, this study identified TRβ to be up-regulated in BRCA1 associated breast cancer and revealed TRs to be associated with patients’ prognosis. TRs were also found to be expressed in triple negative BRCA1 associated breast cancer. Further studies need to be done in order to evaluate whether TRs may become interesting targets of endocrine therapeutic approaches, especially when tumors are triple-negative.

Adenosine monophosphate activated protein kinase (AMPK), a mediator of estradiol-induced apoptosis in long-term estrogen deprived breast cancer cells.

Estrogens stimulate growth of hormone-dependent breast cancer but paradoxically induce tumor regress under certain circumstances. We have shown that long-term estrogen deprivation (LTED) enhances the sensitivity of hormone dependent breast cancer cells to estradiol (E2) so that physiological concentrations of estradiol induce apoptosis in these cells. E2-induced apoptosis involve both intrinsic and extrinsic pathways but precise mechanisms remain unclear. We found that exposure of LTED MCF-7 cells to E2 activated AMP activated protein kinase (AMPK). In contrast, E2 inhibited AMPK activation in wild type MCF-7 cells where E2 prevents apoptosis. As a result of AMPK activation, the transcriptional activity of FoxO3, a downstream factor of AMPK, was up-regulated in E2 treatment of LTED. Increased activity of FoxO3 was demonstrated by up-regulation of three FoxO3 target genes, Bim, Fas ligand (FasL), and Gadd45α. Among them, Bim and FasL mediate intrinsic and extrinsic apoptosis respectively and Gadd45α causes cell cycle arrest at the G2/M phase. To further confirm the role of AMPK in apoptosis, we used AMPK activator AICAR in wild type MCF-7 cells and examined apoptosis, proliferation and expression of Bim, FasL, and Gadd45α. The effects of AICAR on these parameters recapitulated those observed in E2-treated LTED cells. Activation of AMPK by AICAR also increased expression of Bax in MCF-7 cells and its localization to mitochondria, which is a required process for apoptosis. These results reveal that AMPK is an important factor mediating E2-induced apoptosis in LTED cells, which is implicative of therapeutic potential for relapsing breast cancer after hormone therapy.

In-channel printing-device opening assay for micropatterning multiple cells and gene analysis.

Herein we report an easy but versatile method for patterning different cells on a single substrate by using a microfluidic approach that allows not only spatial and temporal control of multiple microenvironments but also retrieval of specific treated cells to profile their expressed genetic information at around 10-cell resolution. By taking advantages of increased surface area of gold nanoparticles on a poly(dimethylsiloxane) (PDMS) coated substrate, cell adhesive-promotive protein, human fibronectin (hFN) can be significantly accumulated on designed regions where cells can recognize the protein and spread out. Moreover, the whole device can be easily opened by hand without any loss of patterned cells which could be retrieved by mouth-pipet. Consequently, we demonstrate the possibility of analyzing the difference of gene expression patterns between wild type MCF-7 cell and MCF/Adr (drug-resistant cell line) from less than 400 cells in total for a single comprehensive assay, including parallel experiments, controls, and multiple dose treatments. Certain genes, especially the P-glycoprotein coding gene (ABCB1), show high expression level in resistant cells compared with the wild type, suggesting a possible pathway that may contribute to the antidrug mechanism.

Generation of MCF-7 cells with aggressive metastatic potential in vitro and in vivo.

Epithelial-mesenchymal transition (EMT) is a cellular development program characterized by loss of cell adhesion and increased cell mobility. It is essential for numerous processes including metastasis. In this study we have generated "aggressive" MCF-7 breast cancer cells (MCF-7-EMT), which show significantly increased invasion in contrast to wild type MCF-7 (MCF-7 WT) cells. In addition, we have analyzed, whether these cell lines differ in their metastatic behavior in vivo and in expression of invasion and/or EMT-relevant genes. Invasive behavior of different human breast cancer cell lines was tested. "Aggressive" MCF-7 cells (MCF-7-EMT) were generated using coculture and mammosphere culture techniques. To analyze whether or not MCF-7-EMT cells in contrast to MCF-7 WT cells form metastases in vivo, we assessed metastases in a nude mouse model. mRNA expression profiles of MCF-7 WT cells and MCF-7-EMT cells were compared using the Affymetrix micro array technique. Expression of selected genes was validated using real-time PCR. In addition, protein expression of epithelial marker E-cadherin (CDH1) and mesenchymal markers N-cadherin (CDH2), Vimentin (VIM), and TWIST was compared. The breast cancer cell lines showed different invasive behavior from hardly any invasion to a stronger cell movement. Coculture with osteoblast-like MG63 cells led to significantly increased cell invasion rates. The highest increase was shown using MCF-7 WT cells. Generated MCF-7-EMT cells showed significantly increased invasion as compared to MCF-7 WT cells. In 8 of 10 mice bearing orthotopically growing MCF-7-EMT tumors, we could detect metastases in liver and lung. In mice bearing MCF-7 WT tumors (n=10), no metastases were found. MCF-7 WT cells and MCF-7-EMT cells were different in expression of 325 genes. Forty-four of the most regulated 50 invasion and/or EMT-related genes were upregulated and 6 genes were downregulated in MCF-7-EMT cells. Protein expression of mesenchymal markers CDH2, VIM, and TWIST was clearly increased in MCF-7-EMT cells. Protein expression of epithelial marker CDH1 was clearly decreased. With the breast cancer cell lines, MCF-7-EMT and MCF-7 WT cells, we have an excellent model of cells for further studies of EMT and invasion in vitro and in vivo.

Methylation-regulated miR-149 modulates chemoresistance by targeting GlcNAc N-deacetylase/N-sulfotransferase-1 in human breast cancer.

Dysregulation of microRNA is strongly implicated in the chemoresistance of cancer. In this study, we found that miR-149 was downregulated and involved in chemoresistance in adriamycin (ADM)-resistant human breast cancer cells (MCF-7/ADM). Downregulation of miR-149 was related to hypermethylation of its 5'-UTR; this methylation also affected the expression of the glypican 1 gene, which is both the host and the target gene of miR-149. Furthermore, we found that miR-149 modulated chemoresistance through targeting the expression of GlcNAc N-deacetylase/N-sulfotransferase-1 (NDST1). With downregulated miR-149, NDST1 expression was increased in chemoresistant MCF-7/ADM cells versus control MCF-7 wild-type cells. The increased NDST1 then activated a heparan sulfate-related pathway involving activation of heparanase. Finally, expression of miR-149 and NDST1 was confirmed in clinical chemoresistant samples of breast cancers receiving anthracycline/taxane-based chemotherapies. The high expression of NDST1 was also an unfavorable predictor for distant relapse-free survival in Her2 and basal breast cancers. Taken together, our findings demonstrate that miR-149 is regulated by methylation, and is a modulator of cancer chemoresistance by targeting NDST1.