Transformation Of MCF10A Cells Research Articles

Abstract 5365: Zeranol-containing serum (Z-Sera) harvested from 30-day Z-implanted heifers induce transformation of human normal breast epithelial cells through down-regulation of the protein tyrosine phosphatase γ (PTPγ) tumor suppressor gene

Abstract The morbidity of breast cancer ranks first in the U.S. it remains the second leading cause of maligancy-related death in women, regardless of advances in novel therapeutic strategies. Many risk factors have been associated with breast cancer initiation, promotion and progression. Recently, Zeranol (Z), one of six growth promoters approved by FDA for use in the beef industry may cause health concerns due to the consumption of beef products containing bio-active Z metabolites. Our laboratory previously demonstrated that Z down-regulates the expression of estrogen-regulated PTPγ in primary cultured human normal breast epithelial cells and transforms the normal human breast epithelial cell line, MCF-10A, to neoplastic breast cancer cells. In the current study, we investigated the biologically active Z metabolites present in Z-Sera harvested from 30-day post Z implanted heifers (72 mg pellet) on MCF-7 cell growth and PTPγ mRNA expression in MCF-10A cells using non radioactive cell proliferation and real time PCR. Our results showed that Z-Sera at 0.5, 2.5 and 12.5% significantly increased the growth of MCF-7 cells by 42, 71, and 108%, respectively. While the sera harvested from the control heifers did not increase MCF-7 cell growth. Treatment with 2.5% Z-sera harvested 30, 60, 90 days post Z-implantation significantly down-regulated the expression of PTP γ mRNA in MCF-7 cells to 18.2, 36.5 and 51%, respectively. Furthermore, neoplastic transformation of MCF-10A cells resulted after exposure of 2.5% Z-Sera in culture medium to MCF-10A for 21-day. The expression of PTPγ mRNA, in transformed MCF-10A cells, was significantly reduced by 83, 96 and 97%, as compared to the controls. We hypothesize that the transformation of MCF-10A cells by bio-active Z metabolites contained in 2.5% Z-sera may be mediated through the down-regulation of estrogen-regulated PTPγ in MCF-10A cells. We have reported that the growth rate of pre-adipocytes isolated from the beef cattle 30-day post Z-implantation was 12 fold faster than the pre-adipocytes isolated from of the control beef cattle. In summary, our in vitro data suggest that potential adverse health risk may result from consuming beef products containing bio-active Z metabolites. (Supported by NIH grant R01 ES 015212). 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 5365.

Reduced cell death, invasive and angiogenic features conferred by BRCA1-deficiency in mammary epithelial cells transformed with H-Ras

To investigate the role of tumor suppressors BRCA1 and p53 proteins in human breast tumorigenesis, we transformed immortalized human mammary epithelial cells, MCF10A, with or without BRCA1/p53 gene-specific knockdowns. Stable knockdown of BRCA1 alone in MCF10A cells led to centrosome amplification, impaired p53 protein stability, increased sensitivity towards DNA-damaging agents, defective chromosomal condensation at mitosis and elevated protein levels of cyclin D1 and c-myc. While over-expression of mutant H-Ras transformed MCF10A cells, depletion of BRCA1 dramatically enhanced the in vivo tumorigenesis that was associated with higher levels of VEGF, enhanced vascularization and less apoptosis in the BRCA1-deficient Ras-transformed tumors. The Ras-transformed BRCA1-deficient tumors exhibited features of the epithelial-to-mesenchymal transition, appeared to secrete matrix metalloproteases as visualized by in vivo bio-imaging of tumors using fluorescent probe MMP680, and were locally metastatic to lymph nodes. Our results suggest that loss of BRCA1 pathway function may contribute to the aggressiveness of Ras-MAPK driven human breast cancer with associated increase in levels of cyclin D1 and c-myc, enhanced MAPK activity, angiogenic potential & invasiveness. This mammary xenograft tumor model may be useful as a tool to understand human breast tumor angiogenesis and metastasis, as well as to test candidate therapeutics.

Cadmium and Breast Cancer: Exposure Associated with Basal-Like Phenotype

Cadmium has been linked with several human diseases including chronic kidney disease and cancer. As a carcinogen, cadmium targets several sites that are considered endocrine-sensitive, and some data suggest the breast may be among them. Although cadmium has been hypothesized to act as a metalloestrogen—a metal that triggers an estrogen-like reaction—research to date has not confirmed this as a mechanism linking cadmium and breast cancer. Additionally, although many breast cancers are estrogen-dependent, some of the most difficult-to-treat cases are not. A new study finds that cadmium can induce malignant transformation in breast cells in vitro regardless of the absence of estrogen receptors, strengthening evidence that cadmium exposure may be a factor in breast cancer, a leading cause of cancer deaths among women [EHP 117:1847–1852; Benbrahim-Tallaa et al.]. MCF-10A cells, which are derived from normal human breast epithelium, were grown with either no cadmium exposure or continuous cadmium exposure (2.5 μM) for up to 40 weeks. Positive controls included MCF-7 human breast cancer cells (which express the estrogen receptors ER-α and ER-β) and SKBR3 breast cancer cells (which express HER2, a receptor that can be overexpressed in certain malignant breast cancer cells). In contrast, MCF-10A cells do not express ER-α, ER-β, or HER2 proteins, although expression can be acquired in carcinogenesis. Chronic cadmium exposure of the MCF-10A cells yielded increased expression of matrix metalloproteinase-9, an enzyme that facilitates tumor cell invasion. These cells also formed cell mounds, indicating a loss of contact inhibition (the natural process of cell growth stopping once a certain density of cells is reached). When these transformed cells were implanted in mice, they formed highly aggressive tumors that demonstrated metastatic potential. Transformed MCF-10A cells remained negative for ER-α and ER-β and also lacked HER2 protein. However, metallothionein, typically overexpressed in ER-negative breast cancers, was elevated as were several other breast cancer markers. These characteristics collectively suggest that cadmium could be a risk factor for a basal-like breast cancer phenotype, which is clinically associated with a higher risk of relapse after treatment and lower survival rates. The precise mechanism by which cadmium may transform breast cells is unknown, but the results of this study suggest it is unlikely to be a metalloestrogenic effect via estrogen receptors. Although additional research is needed to define the mechanism, the current study provides strong evidence that cadmium may play a role in human breast cancer.

Increasingly transformed MCF-10A cells have a progressively tumor-like phenotype in three-dimensional basement membrane culture

BackgroundMCF-10A cells are near diploid and normal human mammary epithelial cells. In three-dimensional reconstituted basement membrane culture, they undergo a well-defined program of proliferation, differentiation, and growth arrest, forming acinar structures that recapitulate many aspects of mammary architecture in vivo. The pre-malignant MCF-10AT cells and malignant MCF-10CA1a lines were sequentially derived from the MCF-10A parental cell line first by expression of a constitutively active T24 H-Ras generating the MCF-10AT cell line. This was followed by repeated selection for increasingly aggressive tumor formation from cells recovered from xenograft tumors in immuno-compromised mice, generating the MCF-10CA1a cell line. When inoculated subcutaneously into the flanks of immuno-compromised mice, MCF-10AT cells occasionally form tumors, whereas MCF-10CA1a cells invariably form tumors with a shorter latency than MCF-10AT derived tumors.ResultsMCF-10AT cells grown in three-dimensional basement membrane culture form complex multi-acinar structures that produce a basement membrane but undergo delayed cell cycle arrest and have incomplete luminal development. MCF-10CA1a cells grown in three-dimensional basement membrane culture form large, hyper-proliferative masses, that retain few characteristics of MCF10A acini and more closely resemble tumors.ConclusionHere we report on the growth and differentiation properties of these three matched cell lines in three-dimensional basement membrane culture. Features of tissue morphogenesis were assessed, including proliferation, basement membrane formation, polarization of alpha-6 beta-4 integrin to the basement membrane, formation of cell:cell junctions, and apoptosis for luminal clearance. The matched series of normal MCF-10A, pre-malignant MCF-10AT, and malignant MCF-10CA1a cells offers a unique opportunity to study the mechanisms of malignant progression both in a three-dimensional microenvironment and in the same cell background.

Identification of Transformation-Related Pathways in a Breast Epithelial Cell Model Using a Ribonomics Approach

The aberrant expression of many genes is a common feature in the malignant transformation of cells. In mammalian cells, posttranscriptional gene regulatory processes are emerging as critical determinants controlling gene expression both in physiologic and pathologic conditions. These regulatory mechanisms are directed primarily by the interaction of mRNAs with specific RNA-binding proteins (RBP). There is an emerging body of data demonstrating that two RBPs, AUF1 and HuR, can antagonistically affect the posttranscriptional fate of target mRNAs, as well as concurrently bind to common target transcripts. Employing MCT-1 oncogene-mediated transformation of immortalized breast epithelial MCF10A cells, we characterized the largely reciprocal association of these two RBPs with target mRNAs and their influence on protein expression vis-a-vis cellular transformation. Using a ribonomics approach, we identified mRNAs from cancer-related pathways whose association with AUF1 and/or HuR were altered when comparing immortalized with transformed MCF10A cells. Significantly, we were able to show that knockdown of HuR expression using RNA interference reduced anchorage-independent growth capacity in transformed MCF10A cells and decreased protein expression of a number of validated target genes. Our data show that the global alterations in binding of HuR and AUF1 with target transcripts have a critical role in posttranscriptional regulation of genes encoding proteins involved in breast epithelial cell transformation. These findings further support the feasibility of using a ribonomics approach for the identification of cancer-related pathways.

Transformation of MCF-10A cells by random mutagenesis with frameshift mutagen ICR191: A model for identifying candidate breast-tumor suppressors

BackgroundWidely accepted somatic mutation theory of carcinogenesis states that mutations in oncogenes and tumor suppressor genes in genomes of somatic cells is the cause of neoplastic transformation. Identifying frequent mutations in cancer cells suggests the involvement of mutant genes in carcinogenesis.ResultsTo develop an in vitro model for the analysis of genetic alterations associated with breast carcinogenesis, we used random mutagenesis and selection of human non-tumorigenic immortalized breast epithelial cells MCF-10A in tissue-culture conditions that mimic tumor environment. Random mutations were generated in MCF-10A cells by cultivating them in a tissue-culture medium containing the frameshift-inducing agent ICR191. The first selective condition we used to transform MCF1-10A cells was cultivation in a medium containing mutagen at a concentration that allowed cell replication despite p53 protein accumulation induced by mutagen treatment. The second step of selection was either cell cultivation in a medium with reduced growth-factor supply or in a medium that mimics a hypoxia condition or growing in soft agar. Using mutagenesis and selection, we have generated several independently derived cultures with various degrees of transformation. Gene Identification by Nonsense-mediated mRNA decay Inhibition (GINI) analysis has identified the ICR191-induced frameshift mutations in the TP53, smoothelin, Ras association (RalGDS/AF-6) domain family 6 (RASSF6) and other genes in the transformed MCF-10A cells. The TP53 gene mutations resulting in the loss of protein expression had been found in all independently transformed MCF-10A cultures, which form large progressively growing tumors with sustained angiogenesis in nude mice.ConclusionIdentifying genes containing bi-allelic ICR191-induced frameshift mutations in the transformed MCF-10A cells generated by random mutagenesis and selection indicates putative breast-tumor suppressors. This can provide a model for studying the role of mutant genes in breast carcinogenesis.

Myosin light chain kinase plays a role in the regulation of epithelial cell survival

Myosin II activation is essential for stress fiber and focal adhesion formation, and is implicated in integrin-mediated signaling events. In this study we investigated the role of acto-myosin contractility, and its main regulators, i.e. myosin light chain kinase (MLCK) and Rho-kinase (ROCK) in cell survival in normal and Ras-transformed MCF-10A epithelial cells. Treatment of cells with pharmacological inhibitors of MLCK (ML-7 and ML-9), or expression of dominant-negative MLCK, led to apoptosis in normal and transformed MCF-10A cells. By contrast, treatment of cells with a ROCK inhibitor (Y-27632) did not induce apoptosis in these cells. Apoptosis following inhibition of myosin II activation by MLCK is probably meditated through the death receptor pathway because expression of dominant-negative FADD blocked apoptosis. The apoptosis observed after MLCK inhibition is rescued by pre-treatment of cells with integrin-activating antibodies. In addition, this rescue of apoptosis is dependent on FAK activity, suggesting the participation of an integrin-dependent signaling pathway. These studies demonstrate a newly discovered role for MLCK in the generation of pro-survival signals in both untransformed and transformed epithelial cells and supports previous work suggesting distinct cellular roles for Rho-kinase- and MLCK-dependent regulation of myosin II.

Breast cancer progression in MCF10A series of cell lines is associated with alterations in retinoic acid and retinoid X receptors and with differential response to retinoids.

In most breast carcinomas and in breast cancer cell lines, retinoic acid receptor beta (RARbeta) is lost or down-regulated, whereas retinoic acid receptor alpha and gamma (RARalpha, gamma) and retinoid X receptors (RXRalpha, beta, gamma) are variably expressed. Little is known about alterations of the above receptors in hyperplastic and premalignant stages of breast cancer development. In this study, we employed the MCF10A series of breast epithelial cell lines (the parental and benign MCF10A, premalignant MCF10AT, and malignant MCF10CA1a) to assess whether in the course of their malignant transformation specific alterations in RARalpha, beta, gamma and RXRalpha, beta, gamma expression occur and whether they may affect the sensitivity of cells to retinoids. Malignant properties of the above cell lines were estimated by the nude mice xenograft transplantation assay. Among the above receptors most significant alterations occurred in RARbeta2, which was detected in the normal breast epithelial cells both, at mRNA and protein levels, but expressed in the MCF10A cell lines at mRNA level only. The transformation of benign MCF10A cells into premalignant MCF10AT and malignant MCF10CA1a was also associated with increase in RARalpha, RARgamma, RXRalpha, and RXRbeta proteins expression. All-trans retinoic acid (atRA), 9-cis retinoic acid (9cRA), and 4-(hydroxyphenyl) retinamide (4-HPR) induced RARbeta2 protein expression exclusively in the benign MCF10A cells and the former two retinoids, mRNA expression in MCF10A and MCF10AT cells, but not in malignant, MCF10CA1a cells, suggesting that the loss of inducible RARbeta expression is associated with the progression and malignant transformation of MCF10A cells. Retinoids also variable decreased the RARalpha, RARgamma and RXRalpha protein expression preferentially in the premalignant and malignant, but not in benign MCF10A cells. Among the above retinoids, 4-HPR was most efficacious in inhibiting the growth of the three cell lines and this apparently was not dependent on the levels of the RARbeta2 transcriptional activation. Thus, our data support the hypothesis that breast epithelial cells in the course of their progression and malignant transformation may differentially respond to retinoids and that not only RARbeta, but RARalpha, gamma and/or RXRalpha, beta may also serve as potential targets for retinoids in breast cancer prevention and therapy trials.

Cigarette smoke condensate-induced transformation of normal human breast epithelial cells in vitro.

In the present study, we showed that a single-dose treatment of normal breast epithelial cell line, MCF10A, for 72 h with cigarette smoke condensate (CSC) resulted in a transformed phenotype. The anchorage-dependent growth of these cells was decreased due to increased cell cycle arrest in S-G2/M phase; however, the surviving cells developed resistance due to an increased Bcl-xL to Bax ratio. Levels of PCNA and gadd45 proteins--involved in DNA repair in response to genomic damage--were increased, suggesting that the cells were responding to CSC-induced genomic damage. The transformation of MCF10A cells was determined by their colony-forming efficiency in soft-agar in an anchorage-independent manner. CSC-treated MCF10A cells efficiently formed colonies in soft-agar. We then re-established cell lines from the soft-agar colonies and further examined the persistence of their transforming characteristics. The re-established cell lines, when plated after 17 passages without CSC treatment, still formed colonies in the soft-agar. An increased staining of neuropilin-1 (NRP-1) further showed a transformation characteristic of MCF10A cells treated with CSC. In summary, our results suggest that CSC is capable of transforming the MCF10A cells in vitro, supporting the role of cigarette smoking and increased risk for breast cancer.

Activation of Fatty Acid Synthesis during Neoplastic Transformation: Role of Mitogen-Activated Protein Kinase and Phosphatidylinositol 3-Kinase

Activation of fatty acid synthase (FAS) expression and fatty acid synthesis is a common event in tumor cells from a variety of human cancers and is closely linked to malignant transformation and to tumor virulence in population studies of human cancer. We now show that, in contrast to nutritional regulation of lipogenesis in liver or adipose tissue, changes in fatty acid metabolism during in vitro transformation of the human mammary epithelial cell line MCF-10a are driven by increases in epidermal growth factor signaling, acting in major part through the mitogen-activated protein (MAP) kinase and phosphatidylinositol (PI) 3-kinase signaling cascades. H- ras transformation of MCF-10a cells resulted in upregulation of MAP kinase and PI 3-kinase signals, upregulation of sterol regulatory element binding protein 1 (SREBP-1) transcription factor levels, and upregulation of FAS expression and FA synthesis. Deletion of the major SREBP binding site from the FAS promoter abrogated transcription in transformed MCF-10a cells. Inhibitors of MAP and PI 3-kinases downregulated SREBP-1 levels and decreased transcription from the FAS promoter, reducing FAS expression and fatty acid synthesis in transformed MCF-10a cells and in MCF-7 and HCT116 carcinoma cells. H- ras transformation sensitized MCF-10a cells to the FAS inhibitors cerulenin and C-75. These results confirm an important role for SREBP-1 in neoplastic lipogenesis, and provide a likely basis for the linkage of upregulated fatty acid metabolism with neoplastic transformation and with tumor virulence, since MAP and PI 3-kinase signaling contributes to both.

Defective repression of c-myc in breast cancer cells: A loss at the core of the transforming growth factor beta growth arrest program.

Loss of growth inhibitory responses to the cytokine transforming growth factor beta (TGF-beta) in cancer cells may result from mutational inactivation of TGF-beta receptors or their signal transducers, the Smad transcription factors. In breast cancer, however, loss of TGF-beta growth inhibition often occurs without a loss of these signaling components. A genome-wide analysis of rapid TGF-beta gene responses in MCF-10A human mammary epithelial cells and MDA-MB-231 breast cancer cells shows that c-myc repression, a response that is key to the TGF-beta program of cell cycle arrest, is selectively lost in the cancer cell line. Transformation of MCF-10A cells with c-Ha-ras and c-erbB2 oncogenes also led to a selective loss of c-myc repression and cell cycle arrest response. TGF-beta stimulation of epithelial cells rapidly induces the formation of a Smad complex that specifically recognizes a TGF-beta inhibitory element in the c-myc promoter. Formation of this complex is deficient in the oncogenically transformed breast cells. These results suggest that a Smad complex that specifically mediates c-myc repression is a target of oncogenic signals in breast cancer.

Regulation of heparin-binding EGF-like growth factor expression in Ha-ras transformed human mammary epithelial cells.

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) mRNA and protein expression is induced by EGF in MCF-10A nontransformed and Ha-ras transfected human mammary epithelial cells. The anti-EGF receptor (EGFR) blocking monoclonal antibody (MAb) 225 and the EGFR tyrosine kinase inhibitor PD153035 were able to inhibit the induction of HB-EGF mRNA levels in MCF-10A cells. However, the Ha-ras transformed MCF-10A cells were more refractory to inhibition by these agents and only a combination of the 225 MAb and PD153035 was able to significantly abrogate HB-EGF induction by EGF. The anti-erbB2 MAb L26 which interferes with heterodimer formation was able to block HB-EGF induction in response to EGF in MCF-10A cells and in the Ha-ras transformed cells only when used in combination with either the 225 MAb or PD153035. The MEK inhibitor PD90859 completely blocked EGF induction of HB-EGF mRNA levels in the nontransformed and Ha-ras transformed MCF-10A cells, which indicates that MAPK is involved in the signaling pathway of HB-EGF induction by EGF. An increase in the levels of HB-EGF may, therefore, be an important contributor to oncogenic transformation that is caused by Ha-ras overexpression in mammary epithelial cells. J. Cell. Physiol. 186:233-242, 2001. Published 2001 Wiley-Liss, Inc.

RAS transformation causes sustained activation of epidermal growth factor receptor and elevation of mitogen-activated protein kinase in human mammary epithelial cells.

Activation of the ras oncogene is an important step in carcinogenesis. Human MCF-10A mammary epithelial cells were transformed with a point-mutated form of the Ha-ras oncogene. Epidermal growth factor receptor (EGFR) phosphorylation levels were chronically elevated after EGF induction and the EGFR ligand-driven internalization rate was slower in Ha-ras transformed MCF-10A cells. Additionally, basal levels of p42/44 mitogen-activated protein kinase (MAPK) expression and enzyme activity were significantly higher in Ha-ras transformed cells, localized predominantly in the nucleus. The anti-EGFR monoclonal antibody (MAb) 225 and the EGFR tyrosine kinase inhibitor PD153035 blocked anchorage-independent growth of Ha-ras transformed cells in soft agar and were more effective when used in combination. The MEK inhibitor PD98059 and anti-erbB-2 MAb L26 also suppressed colony formation of Ha-ras transformed cells in soft agar. Therefore, Ha-ras transformation leads to an augmentation in signaling through the EGFR as a result of an increase in ligand-dependent phosphorylation, a decrease in its internalization and an up-regulation in basal p44/42 MAPK levels. These effects may contribute to uncontrolled growth of Ha-ras-transformed human mammary epithelial cells.

Enhanced expression of heregulin in c-erb B-2 and c-Ha-ras transformed mouse and human mammary epithelial cells

Heregulin beta 1 was found to stimulate the anchorage-dependent, serum-free growth of nontransformed human MCF-10A mammary epithelial cells. Unlike epidermal growth factor, transforming growth factor alpha, or amphiregulin, heregulin beta 1 was also able to induce the anchorage-independent growth of MCF-10A cells. In contrast, the anchorage-independent, serum-free growth of c-Ha-ras or c-erb B-2 transformed MCF-10A cells was unaffected by heregulin beta 1, whereas heregulin beta 1 was able to stimulate the anchorage-independent growth of these cells. c-Ha-ras or c-erb B-2 (c-neu) transformed MCF-10A or mouse NOG-8 mammary epithelial cells express elevated levels of 2.5, 5.0, 6.5, 6.8, and 8.5 kb heregulin mRNA transcripts and/or synthesize cell-associated 25, 29, 50, and 115 kDa isoforms of heregulin. Since the MCF-10A cells and transformants also express c-erb B-3, these data suggest that endogenous heregulin might function as an autocrine growth factor for Ha-ras or erb B-2 transformed mammary epithelial cells.

THE EFFECT OF FGF-3 INT-2 ON GROWTH AND TRANSFORMATION OF MCF-10A NORMAL HUMAN MAMMARY EPITHELIAL-CELLS IS DISTINCT FROM FGF-1 AND FGF-2

Fibroblast growth factors (FGFs) are related polypeptides with mitogenic activity on cells of mesodermal and neuroectodermal origin. The Fgf-3 gene shares high homology with FGF-2 and its protein product can substitute FGF-2 as a growth factor. Other observations, however, indicate that Fgf-3 has specialized functions. We have investigated the effect of the expression and secretion of Fgf-3 on the growth and transformation of the human breast epithelial cell line MCF-10A. Overexpression of Fgf-3 stimulates proliferation of these cells in serum-free medium and induces anchorage-independent colony formation in soft agar. In contrast, these effects were not observed with purified FGF-1 and FGF-2 on either the parental or the Fgf-3-MCF-10A cells. Thus, Fgf-3 is distinct from FGF-1 and FGF-2 for its ability to induce cell proliferation and transformation of MCF-10A cells. This difference could be due, at least in part, to the expression of a specific set of FGF receptors with higher affinity for FGF-3 than FGF-1 or FGF-2.