Malignant Human Breast Epithelial Cells Research Articles

Drug repositioning for immunotherapy in breast cancer using single-cell analysis.

Immunomodulatory peptides, while exhibiting potential antimicrobial, antifungal, and/or antiviral properties, can play a role in stimulating or suppressing the immune system, especially in pathological conditions like breast cancer (BC). Thus, deregulation of these peptides may serve as an immunotherapeutic strategy to enhance the immune response. In this meta-analysis, we utilized single-cell RNA sequencing data and known therapeutic peptides to investigate the deregulation of these peptides in malignant versus normal human breast epithelial cells. We corroborated our findings at the chromatin level using ATAC-seq. Additionally, we assessed the protein levels in various BC cell lines. Moreover, our in-house drug repositioning approach was employed to identify potential drugs that could positively impact the relapse-free survival of BC patients. Considering significantly deregulated therapeutic peptides and their role in BC pathology, our approach aims to downregulate B2M and SLPI, while upregulating PIGR, DEFB1, LTF, CLU, S100A7, and SCGB2A1 in BC epithelial cells through our drug repositioning pipeline. Leveraging the LINCS L1000 database, we propose BRD-A06641369 for B2M downregulation and ST-4070043 and BRD-K97926541 for SLPI downregulation without negatively affecting the MHC complex as a significantly correlated pathway with these two genes. Furthermore, we have compiled a comprehensive list of drugs for the upregulation of other selected immunomodulatory peptides. Employing an immunotherapeutic approach by integrating our drug repositioning pipeline with single-cell analysis, we proposed potential drugs and drug targets to fortify the immune system against BC.

GP5 regulates epithelial-mesenchymal transition in breast cancer via the PI3K/AKT signaling pathway.

Recent evidences have shown that glycoprotein V (GP5) protein, which is initially considered as an important adhesion molecule unique to the megakaryocyte line, was also specifically expressed in malignant human breast epithelial cells. However, its expression level and function are not clear. This study aimed to reveal the abnormal expression of GP5 in breast cancer (BC), research the associations between the GP5 abnormal expression and BC progression, and explore the molecular mechanism of GP5 in BC. Immunohistochemistry, Western blot (WB), and quantitative reverse transcription-polymerase chain reaction (RT-PCR) assays were used to determine the expression patterns of GP5 in BC tissues and cells. The expression profiles of GP5 in the Cancer Genome Atlas databases were analyzed by UALCAN. The GP5 knockdown and over-expression BC cell lines were constructed and confirmed by RT-PCR and WB. Transcriptome sequencing and KEGG database were performed to screen cellular processes and signal pathways. Phosphatidylinositol 3-kinase (PI3K)/AKT pathway was verified by RT-qPCR, and epithelial-mesenchymal transition (EMT) was confirmed by WB. The results indicated GP5 was highly expressed in BC tissues and might play an important role as a cancer-promoting gene in BC. The high expression of GP5 was significantly associated with higher nuclear grade, higher TNM stage, and human epidermal growth factor receptor 2 (HER2) negativity. GP5 may promote the proliferation, invasion, and metastasis of BC cells by activating PI3K/AKT signaling pathway to up-regulate the EMT. This study provides a new idea that GP5 was expected to become a potential molecular target for early BC clinic diagnosis and treatment.

Abstract 37: Pulmonary pre-metastatic modifications induced by breast cancer

Abstract Breast cancer can be highly curable at early stages, while the survival rate plummets with the occurrence of metastasis. Thus, studies of molecular mechanisms that allow cancer cells to leave the primary tumor and ultimately establish colonies at distant sites are of crucial importance. First proposed by Stephen Paget in 1889, the “seed and soil” hypothesis suggests that the secondary organ microenvironment undergoes constant modification induced by the primary tumor through circulating chemokines. This leads to the migration of activated bone marrow derived cells to secondary organs, increased matrix metalloproteases (MMPs), and extracellular matrix (ECM) modifications in the secondary organ, collectively termed the pre-metastatic niche, which provides desirable docking sites for cancer cells. Our previous studies have shown persistent changes in the collagenous stroma and proteoglycans in pre-metastatic lungs of mice growing metastatic breast tumors, whose extent directly correlated with the malignancy of the primary tumor [1]. Elevated proteolytic MMP activity was also detected in these pre-metastatic lungs [2]. Based on these findings, we have continued our pre-metastatic niche studies by building an appropriate cell culture model, which consists of lung fibroblasts that were exposed to cell culture media conditioned by differentially malignant human breast epithelial cells. Cell lines used for fibroblast conditioning were nonmalignant MCF-12A breast epithelial cells (control), non-metastatic MCF-7 breast cancer cells, and highly metastatic, triple-negative MDA-MB-231 breast cancer cells. We focused our studies on ECM- and MMP-related changes, which we analyzed using qRT-PCR and immunoblotting. qRT-PCR analysis revealed that metastatic MDA-MB-231 cells induced an increase in the ECM protein Col14A1 as well as MMP-2 and MMP-14 in lung fibroblast as compared to induction by MCF-12A control. Immunoblotting analyses of lung fibroblasts showed that breast cancer cells induced increases in MMP expression in lung fibroblasts. Even higher MMP levels in fibroblasts were induced by metastatic MDA-MB-231 cells as compared to non-metastatic MCF-7 cells. Breast cancer cells also induced decreased expression levels of secreted MMPs in lung fibroblasts, most likely due to an enhanced release of these MMPs. This decrease was most significantly induced by the metastatic cell line. In conclusion, we have observed elevated levels in ECM molecules and MMPs in lung fibroblasts upon exposure of conditioned media from breast cancer cells. Our findings point towards an important role of lung fibroblasts in the evolution of the pre-metastatic niche in modifying the ECM, and their ability to directly respond to factors released from primary breast cancer cells with which they are not in direct contact. [1] Paidi SK et al. Cancer Res. 2017; 77:247-56. [2] Rizwan et al. Npj Breast Cancer 2015; 1: 15017. Citation Format: Ruoqing Cai, Caitlin M. Tressler, Menglin Cheng, Kanchan Sonkar, Santosh K. Paidi, Ishan Barman, Kristine Glunde. Pulmonary pre-metastatic modifications induced by 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 37.

Epigenetic changes in cancer by Raman imaging, fluorescence imaging, AFM and scanning near-field optical microscopy (SNOM). Acetylation in normal and human cancer breast cells MCF10A, MCF7 and MDA-MB-231.

This paper examines epigenetic changes in breast cancer by Raman imaging, fluorescence imaging, AFM and SNOM and discusses how they contribute to different aspects of tumourigenesis in malignant human breast epithelial cell lines MCF7 and MDA-MB-231 compared with non-malignant MCF10A cell lines. The paper focuses on information that can be extracted from Raman microscopy and Raman imaging for the biological material of nucleoli contained within the cell nucleus and lipid droplets within the cell cytoplasm. The biochemical composition of the nuclei and lipid droplets in the non-malignant and malignant human breast epithelial cell lines has been monitored. The potential of Raman microspectroscopy to monitor acetylation processes and a prognostic value of Raman biomarkers in breast cancer have been discussed.

S100A14, a Member of the EF-hand Calcium-binding Proteins, Is Overexpressed in Breast Cancer and Acts as a Modulator of HER2 Signaling

HER2 is overexpressed in 20–25% of breast cancers. Overexpression of HER2 is an adverse prognostic factor and correlates with decreased patient survival. HER2 stimulates breast tumorigenesis via a number of intracellular signaling molecules, including PI3K/AKT and MAPK/ERK.S100A14,one member of the S100 protein family, is significantly associated with outcome of breast cancer patients. Here, for the first time, we show that S100A14 and HER2 are coexpressed in invasive breast cancer specimens,andthere is a significant correlation between the expression levels of the two proteins by immunohistochemistry. S100A14 and HER2 are colocalized in plasma membrane of breast cancer tissue cells and breast cancer cell lines BT474 and SK-BR3. We demonstrate that S100A14 binds directly to HER2 by co-immunoprecipitation and pull-down assays. Further study shows that residues 956–1154 of the HER2 intracellular domain and residue 83 of S100A14 are essential for the two proteins binding.Moreover,we observe a decrease of HER2 phosphorylation, downstream signaling, and HER2-stimulated cell proliferation in S100A14-silenced MCF-7, BT474, and SK-BR3 cells. Our findings suggest that S100A14 functions as a modulator of HER2 signaling and provide mechanistic evidence for its role in breast cancer progression.

Differential gene expression of Wnt signaling pathway in benign, premalignant, and malignant human breast epithelial cells

To gain insight into the role of gene expression alterations in breast cancer progression, we conducted a comprehensive gene expression analysis of a series of cell lines derived from MCF10A, which include benign MCF10A cells, premalignant AT, and malignant CA1a tumor cells. We analyzed gene expression variation using the Agilent Human Genome Oligo Microarray with the goal of identifying gene-specific expression change events. In addition to a previously noted overexpression in oncogene MDM2, HRAS, and PCNA, our studies identified overexpression of Wnt signaling pathway in malignant breast cell lines. The Kaplan-Meier plot showed that high c-Myc expression in breast cancer was associated with tumor progression and the patient's poor survival. This study showed that the Wnt pathway has further provided a basis for the development of potential biomarker for breast cancer prognosis.

The hydroxyl functional group of N-(4-hydroxyphenyl)retinamide mediates cellular uptake and cytotoxicity in premalignant and malignant human epithelial cells

In a previous study, we demonstrated that the anticancer synthetic retinoid N-(4-hydroxyphenyl)retinamide (4HPR) redox cycles at the mitochondrial enzyme dihydroorotate dehydrogenase to trigger anomalous reactive oxygen species (ROS) production and attendant apoptosis in transformed human epithelial cells. Furthermore, we speculated that the hydroxyl functional group of 4HPR was required for this pro-oxidant property. In this study, we investigated the role of the hydroxyl functional group in the in vitro cytotoxicity of 4HPR. Using 4HPR, its primary in vivo metabolite N-(4-methoxyphenyl)retinamide (4MPR), and the synthetic derivative N-(4-trifluoromethylphenyl)retinamide (4TPR), we examined the pro-oxidant and apoptotic effects, as well as the cellular uptake, of these three N-(4-substituted-phenyl)retinamides in premalignant and malignant human skin, prostate, and breast epithelial cells. Compared to 4HPR, both 4MPR and 4TPR were ineffective in promoting conspicuous cellular ROS production, mitochondrial disruption, or DNA fragmentation in these transformed cells. Interestingly, both 4MPR and 4TPR were not particularly cell permeative relative to 4HPR in skin or breast epithelial cells, which implied an additional role for the hydroxyl functional group in the cellular uptake of 4HPR. Moreover, the short-term uptake of 4HPR was directly proportional to cell size, but this characteristic, in obvious contrast to cellular bioenergetic status and/or dihydroorotate dehydrogenase expression, was not fundamentally influential in the overall sensitivity to the promotion of cellular ROS production and apoptosis induction by this agent. Together, these results strongly implicate the hydroxyl functional group in the cytotoxic effects of 4HPR.

Hyperglycaemia confers resistance to chemotherapy on breast cancer cells: the role of fatty acid synthase

The prognosis for women with breast cancer is adversely affected by the comorbidities of obesity and diabetes mellitus (DM), which are conditions associated with elevated levels of circulating fatty acids, hyperglycaemia and hyperinsulinaemia. We investigated the effects of exposure of non-malignant and malignant human breast epithelial cells to elevated levels of fatty acids and glucose on their growth, survival and response to chemotherapeutic agents. We found that palmitate induced cell death in the non-malignant cells but not in the malignant cells, which was abrogated through the inhibition of ceramide production and by oleate but not by IGF1. Fatty acid synthase (FAS) is responsible for the de novo synthesis of fatty acids from sugars, and is over-expressed in many epithelial cancers. Abundance of FAS was higher in malignant cells than in non-malignant cells, and was up-regulated by IGF1 in both cell types. IGF-induced growth of non-malignant cells was unaffected by suppression of FAS expression, whereas that of malignant cells was blocked as was their resistance to palmitate-induced cell death. Palmitate did not affect cell proliferation, whereas oleate promoted the growth of non-malignant cells but had the opposite effect, that is, inhibition of IGF1-induced growth of malignant cells. However, when the phosphatidylinositol 3-kinase pathway was inhibited, oleate enhanced IGF1-induced growth in both cell types. Hyperglycaemia conferred resistance on malignant cells, but not on non-malignant cells, to chemotherapy-induced cell death. This resistance was overcome by inhibiting FAS or ceramide production. Understanding the mechanisms involved in the associations between obesity, DM and breast cancer may lead to more effective treatment regimens and new therapeutic targets.

Rapamycin sensitizes Akt inhibition in malignant human breast epithelial cells

Akt and mTOR are therapeutic targets for the treatment of cancer. The effects of inhibiting mTOR, with rapamycin, and Akt, with A-443654, concurrently, on cell morphology, cell proliferation, the cell cycle, and apoptosis were examined using the benign MCF10A and malignant MCF10CA1a human breast epithelial cells. Rapamycin and A-443654 in combination produced the greatest morphological changes and inhibited cell proliferation by G2/M arrest. Rapamycin and A-443654 in combination induced apoptosis at earlier times and at lower A-443654 concentrations in MCF10CA1a tumor cells than in the benign MCF10A cells. Rapamycin and A-443654 increased p53 and p15 INK4B protein levels, decreased anti-apoptotic Bcl-2 levels, and increased Bad levels in the MCF10CA1a tumor cells by ∼5-fold. These results suggest that the combined inhibition of Akt and mTOR may have beneficial therapeutic and safety margin effects.

Abstract 5367: Medroxyprogesterone acetate impedes 5α-dihydrotesterone induced androgen receptor signaling in normal and malignant human breast epithelial cells

Abstract Combined hormone replacement therapy (cHRT) comprised of equine estrogens and the synthetic progestin medroxyprogesterone acetate (MPA) increases breast cancer risk in post-menopausal women [1], via as yet undetermined mechanisms. In addition to the progesterone receptor (PR), MPA binds with high affinity to the androgen receptor (AR), and therefore has the potential to elicit actions distinct to those of natural progesterone, which does not bind AR. We have proposed that MPA increases breast cancer risk by binding to the AR and interfering with the ability of endogenous AR ligands to inhibit proliferation of normal breast epithelial cells [2, 3]. To further investigate this proposal, we examined the effect of MPA on 5α-dihydrotestosterone (DHT)-induced AR signaling in normal human breast tissue and in a breast cancer cell line (ZR75-1) that expresses estrogen receptor alpha (ERα), PR, and AR. Normal human breast tissues was obtained from 12 post-menopausal women following surgery for benign or malignant disease. Normal histology was confirmed by a pathologist. Breast tissues (3mm3) were cultured in triplicate for 48 hours on gelatine sponges soaked in media containing 10% steroid deplete fetal calf serum (FCS) and DHT (1nM), MPA (1nM), or the AR antagonist, bicalutamide (Bic; 1uM), alone and in combination. Following culture, expression of AR, Ki67 (a marker of cell proliferation) and bcl-2 (a pro-survival factor) was assessed by immunohistochemistry. ZR75-1 cells were cultured for 24 and 48 hours in 10% steroid deplete FCS supplemented with DHT, MPA or the combination, followed by immunoblot analysis to detect AR and an androgen-regulated protein, FKBP5. Statistical analysis was preformed by Wilcoxon signed-rank test unless otherwise stated. Normal human breast tissues possessed significantly higher levels of immunoreactive AR than ERα or PR (p<0.0001; t-test for unequal variances). Treatment with DHT significantly increased AR expression (p=0.02) and decreased the percentage of Ki67 positive cells (p=0.03), but had no effect on expression of bcl-2. The effects of DHT on AR and Ki67 were reversed by co-incubation with Bic (p<0.05) or MPA (p<0.05). In ZR75 cells, DHT increased AR protein levels at 24 and 48 hours and increased FKBP5 protein levels at 48 hours. Co-incubation with MPA reversed the effects of DHT on AR and FKBP5 proteins. Collectively, these data support our hypothesis that MPA interferes with the action of endogenous AR ligands in normal and malignant human breast epithelial cells. This interference of androgen action corresponds to an increased proliferation index in normal human breast epithelial cells, which feasibly contributes to an increased risk of developing breast cancer. 1. Chlebowski., R.T., et al., Jama, 2003. 289: 3243-53 2. Birrell, S.N., et al., Faseb J, 2007. 21; 2285-93 3. Peters, A.A., et al., Cancer Res, 2009. 69: 6131-40 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5367.

Abstract B91: RNAi knockdown of Mek2, but not Mek1, increases proliferation of malignant and non-malignant human breast cells

Abstract We are using shRNAs against Mek1 and/or Mek2 to determine the individual contributions of Mek1 and Mek2 to proliferation, migration and Mek inhibitor sensitivity in malignant and non-malignant human breast epithelial cell lines. We found in both malignant (MCF7 and MDAMB231) and non-malignant immortalized (184vTERT and MCF10A) breast cell lines that, despite significant shRNA-mediated reductions (>95%) in the expression of Mek1, Mek2, or both kinases, the cells maintained long-term growth. In fact, the shMek2 lines grew significantly faster and were more motile than controls, suggesting that in some breast cancers, drugs that specifically target Mek1 might be more effective than those which target both Mek1 and 2. In MCF7 cells, levels of phosphorylated (P)-Erk1 and 2 were significantly decreased or undetectable in the presence of Mek shRNAs, indicating that Erk phosphorylation may be dispensable for cell proliferation in these cells. In contrast, in 184vTERT and MCF10A, phosphorylated Erk levels remained constant despite the knockdown of both Mek1 and Mek2. This latter result indicated that, in some breast cells, another kinase is likely to phosphorylate Erk1/2 in the absence of Mek1 and Mek2. The Mek5 kinase is genetically most similar to Mek1 and Mek2, making it an attractive candidate for further investigation. Mek5 is expressed in 184vTERT, and its total protein levels are not altered when Mek1 and 2 are knocked down. A preliminary experiment indicates that siRNA induced knockdown of Mek5 expression leads to decreased ERK1/2 phosphorylation in 184v-TERT cells independently of Mek1/2 expression. We are currently investigating whether this occurs in other breast cell lines. Collectively, our data indicate that in at least some breast cells, both Mek1 and Mek2 are dispensable for proliferation and survival, and that parallel pathways are sufficient for relaying signals from cell surface receptors, independently of Mek1/2 and/or P-Erk1/2. This data may explain why Mek inhibitors, which target both Mek1 and Mek2, have not done well clinically. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B91.

MUC-1-/ESA+ progenitor cells in normal, benign and malignant human breast epithelial cells

The existence of mammary epithelial stem/progenitor cells has been demonstrated in MUC-1-/ESA+ subpopulations of breast epithelial cells. However, knowledge about the expression and localization in benign and malignant breast lesions is unknown. Using a double-staining immunohistochemistry method, we investigated MUC-1-/ESA+ cells in 10 normal breast tissues, 49 cases with fibrocystic disease, 40 fibroadenomas, 36 invasive ductal carcinomas and the breast cancer cell lines MCF-7 and MDA-MB-468. In normal breast tissues MUC-1-/ESA+ cells were mainly found in the suprabasal layer, but under the apical surface of the duct/alveolus. In the hyperplastic areas of fibrocystic disease, the number of this subpopulation of cells was higher than that in hypoplastic areas and in fibroadenomas. In invasive ductal carcinoma, the MMUC-1-/ESA+ cells were heterogeneously present in different carcinoma nests. In the MCF-7 cell line most cells were MUC-1-/ESA+, and in the MDA-MB-468 cell line MUC-1-/ESA+ cells and MUC-1-/ESA+ cells were almost equal. Our results show that the MUC-1-/ESA+ subpopulation increases in fibrocystic disease within the hyperplastic areas, and varies in benign and malignant breast tumours, indicating that breast carcinogenesis may develop from malignant changes of normal MUC-1-/ESA+ cells.

Resveratrol down-regulates the growth and telomerase activity of breast cancer cells in vitro

A number of previous studies investigated the in vitro effects of resveratrol on malignant human breast epithelial cell replication. The aim of the present study was to evaluate the activity of resveratrol on human metastatic breast cancer cells. The study was performed on the MCF-7 tumor cell line. Cell growth, cell cycle perturbation and apoptosis were evaluated by trypan blue dye exclusion assay, flow cytometric analysis and confocal fluorescence microscopy. TRAP assay and Western blot analysis respectively detected levels of telomerase activity and levels of hTERT in intracellular compartments of MCF-7 cells treated with resveratrol. Resveratrol has a direct inhibitory effect on cell proliferation. The results demonstrate that the drug induces apoptosis in MCF-7 cells, in a time- and concentration-related manner. Our results also show that the growth-inhibitory effect of resveratrol on malignant cells is mainly due to its ability to induce S-phase arrest and apoptosis in association with reduced levels of telomerase activity. In particular, TRAP assay and Western blot analysis respectively showed that resveratrol treatment down-regulates the telomerase activity of target cells and the nuclear levels of hTERT, the reverse transcriptase subunit of the telomerase complex. In our experimental model of breast cancer, resveratrol shows direct antiproliferative and pro-apoptotic effects. Studies on telomerase function and intracellular hTERT distribution point out that this agent is endowed with additional suppressive functions on critical tumor biological properties. These results speak in favor of a potential role of resveratrol in chemoprevention/chemotherapy of breast cancer.

The effect of progesterone, testosterone and synthetic progestogens on growth factor- and estradiol-treated human cancerous and benign breast cells

Objective The effects of progesterone (P), testosterone (T), chlormadinone acetate (CMA), medroxyprogesterone acetate (MPA), norethisterone (NET), levonorgestrel (LNG), dienogest (DNG), gestodene (GSD) and 3-ketodesogestrel (KDG) were investigated in normal human breast epithelial MCF10A cells. In addition, the effects of these steroids were tested in estrogen and progesterone receptor positive HCC1500 human primary breast cancer cells. Study design MCF10A cells were incubated with each progestogen at 1 μM and 100 nM for 7 days with growth factors epidermal growth factor (EGF), fibroblast growth factor (FGF) and insulin-like growth factor (IGF-I) (GFs, each 1 pM), and HCC1500 cells with GFs and/or estradiol (E2) 100 pM. Cell proliferation rate was measured by ATP-assay and cell death by photometric enzyme immunoassay. Ratios of cell death:proliferation were calculated. Results MPA and CMA with GFs induced proliferation of MCF10A cells. P, T, NET, LNG, DNG, GSD and KDG had no significant effect. In HCC1500 cells, MPA and CMA with GFs had an inhibitory effect compared to GFs alone. NET, LNG, DNG, GSD, KDG and T enhanced the proliferative effect of GFs. P had no significant effect. No progestogen could further enhance the stimulatory effect of E2 on HCC1500 cells; all but KDG inhibited it. MPA, GSD, T, CMA and NET had an anti-proliferative effect on the mitotic GF and E2 combination. P, LNG, DNG and KDG had no significant effect. Conclusions Estrogens and mitogenic growth factors from stromal breast tissue are significant in growth-regulation of breast cells and may alter responses to progestogens. Certain progestogens are able to induce proliferation of or inhibit growth of benign or malignant human breast epithelial cells independently of the effects of growth factors and E2; therefore, choice of progestogen for hormone therapy may be important in terms of influencing possible breast cancer risk.

RNA Interference–Mediated Choline Kinase Suppression in Breast Cancer Cells Induces Differentiation and Reduces Proliferation

Choline kinase is overexpressed in breast cancer cells and activated by oncogenes and mitogenic signals, making it a potential target for cancer therapy. Here, we have examined, for the first time, the effects of RNA interference (RNAi)-mediated down-regulation of choline kinase in nonmalignant and malignant human breast epithelial cell lines using magnetic resonance spectroscopy (MRS) as well as molecular analyses of proliferation and differentiation markers. RNAi knockdown of choline kinase reduced proliferation, as detected by proliferating cell nuclear antigen and Ki-67 expression, and promoted differentiation, as detected by cytosolic lipid droplet formation and expression of galectin-3. The functional importance of RNAi-mediated choline kinase down-regulation on choline phospholipid metabolism was confirmed by the significant reduction of phosphocholine detected by MRS. These results strongly support the targeting of choline kinase in breast cancer cells with RNAi and show the potential ability of noninvasive MRS to detect and evaluate future treatments incorporating such strategies.

Activation of the MAP kinase pathway induces chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII) expression in human breast cancer cell lines.

Growth factors are essential for cellular growth and differentiation in both normal and malignant human breast epithelial cells. In the present study we investigated the effect of epidermal growth factor (EGF), transforming growth factor alpha (TGFalpha) and phorbol myristate acetate (PMA) on chicken ovalbumin upstream promoter-transcription factor (COUP-TF) expression in human breast cancer cells. The orphan receptors COUP-TFI and COUP-TFII are members of the nuclear receptor superfamily. The high degree of evolutionary conservation of these proteins strongly argues for an important biological function. COUP-TF expression was highest in SK-BR3 cells (approximately 130 amol/ micro g total RNA), while the lowest COUP-TF expression was observed in MCF-7 cells (3.5 amol/ micro g total RNA). While treatment of EGF, TGFalpha and PMA induced expression of COUP-TFII, COUP-TFI did not respond to these agents. Oncostatin M (OSM) is known to exert an antiproliferative effect in breast cancer cells. Treatment of MCF-7 cells with OSM resulted in an approximately 90% reduction of COUP-TFII mRNA expression. In SK-BR3 cells, treatment with the MEK inhibitor UO126 resulted in a profound suppression of endogenous COUP-TFII expression. Furthermore, cotreatment with UO126 prevented induction of COUP-TFII expression by EGF in MCF-7 cells. In conclusion, our data provide evidence, for the first time, that mitogenic substances which activate the MAP kinase pathway, can induce COUP-TFII expression. Our results strongly suggest that an active MAP kinase pathway is essential for COUP-TFII expression in human breast cancer cells.

The effect of oestrogen and tamoxifen on purified normal and malignant human primary breast epithelial cells

The effect of oestrogen and tamoxifen on purified normal and malignant human primary breast epithelial cells

Fas ligand is expressed in normal breast epithelial cells and is frequently up-regulated in breast cancer.

Fas (CD95/Apo-1) is a cell membrane receptor that upon binding by its ligand (FasL), triggers a signal resulting in apoptotic cell death. Fas is produced by breast epithelial cells, but its contribution to breast tissue homeostasis is unknown. This study investigated whether FasL is synthesized in the breast. By reverse transcription-polymerase chain reaction (RT-PCR), western blotting, and immunohistochemistry, FasL expression was analysed in normal and malignant human breast epithelial cell lines, normal breast tissue, benign breast disease (fibrocystic changes, fibroadenoma), and breast cancer (ductal carcinoma in situ; invasive ductal, lobular, mucinous and medullary carcinomas). The results demonstrate expression of FasL by normal breast epithelial cells and show a marked increase of FasL protein in the majority of breast carcinomas, compared with normal breast tissue and benign breast disease. By western blot analysis, soluble FasL was detected in culture supernatants of one of three normal breast epithelial cell lines and in all four breast cancer cell lines tested. The expression of Fas protein was more heterogeneous in benign and malignant breast tissue, with expression levels ranging from weak to strong, but breast cancer cells frequently exhibited a weaker Fas expression than surrounding residual normal breast epithelial cells. In vitro, two out of three normal breast epithelial cell lines were sensitive to cell death induction by an agonistic anti-Fas antibody. Co-treatment with cycloheximide, an inhibitor of protein translation, rendered the resistant cell line sensitive. In contrast, two out of four breast cancer cell lines were resistant to the anti-Fas antibody and this resistance could not be reversed by cycloheximide. These results suggest that increased expression of FasL may confer an advantage on breast cancer cells, possibly by eliminating tumour-infiltrating immune cells, and/or by facilitating tissue destruction during invasion.

Biologic effects of heregulin/neu differentiation factor on normal and malignant human breast and ovarian epithelial cells.

The heregulins are a family of ligands with ability to induce phosphorylation of the p185HER-2/neu receptor. Various investigators have reported a variety of responses of mouse and human breast and ovarian cells to this family of ligands including growth stimulation, growth inhibition, apoptosis and induction of differentiation in cells expressing the HER-2/neu receptor. Some of the disparity in the literature has been attributed to variations in the cell lines studied, ligand dose applied, methodologies utilized or model system evaluated (i.e. in vitro or in vivo). To evaluate the effects of heregulin on normal and malignant human breast and ovarian epithelial cells expressing known levels of the HER-2/neu receptor, this report presents the use of several different assays, performed both in vitro and in vivo, in vitro proliferation assays, direct cell counts, clonogenicity under anchorage-dependent and anchorage-independent conditions, as well as the in vivo effects of heregulin on human cells growing in nude mice to address heregulin activity. Using a total of five different biologic assays in nine different cell lines, across two different epithelia and over a one log heregulin dose range, we obtained results that clearly indicate a growth-stimulatory role for this ligand in human breast and ovarian epithelial cells. We find no evidence that heregulin has any growth-inhibitory effects in human epithelial cells. We also quantitated the amount of each member of the type I receptor tyrosine kinase family (RTK I, i.e. HER-1, HER-2, HER-3 and HER-4) in the cell lines employed and correlated this to their respective heregulin responses. These data demonstrate that HER-2/neu overexpression itself affects the expression of other RTK I members and that cells expressing the highest levels of HER-2/neu have the greatest response to HRG.

Correlation between cell cycle regulators and the immortalization and transformation of human breast epithelial cell lines.

Cellular proliferation, essential for normal development, may result in neoplastic growth when the cell cycle clock is disrupted. In order to determine whether the protein expression of cell cycle regulators differs among normal, immortalized non-tumorigenic and malignant human breast epithelial cells (HBECs), we analyzed the protein expression of cyclins D1, D3, A and E, cyclin-dependent kinase (cdk) 4 and c-fos in exponentially growing MCF-10M, MCF-10F, and MCF-7 cells. The tumorigenicity of HBECs in vivo correlated with both cell cycle regulators and early-gene protein expression in vitro. The differential expression of cyclin E- and cyclin A-related proteins and their putative relevance in the tumorigenic properties of HBECs are also discussed.