Sandra V. Fernandez

17-Beta-estradiol induces transformation and tumorigenesis in human breast epithelial cells

Breast cancer is a malignancy whose dependence on estrogen exposure has long been recognized even though the mechanisms whereby estrogens cause cancer are not clearly understood. This work was performed to determine whether 17β‐estradiol (E2), the predominant circulating ovarian steroid, is carcinogenic in human breast epithelial cells and whether nonreceptor mechanisms are involved in the initiation of breast cancer. For this purpose, the effect of four 24 h alternate periods of 70 nM E2 treatment of the estrogen receptor alpha (ER‐α) negative MCF‐10F cell line on the in vitro expression of neoplastic transformation was evaluated. E2 treatment induced the expression of anchorage‐independent growth, loss of ductulogenesis in collagen, invasiveness in Matrigel, and loss of 9p11–13. Only invasive cells that exhibited a 4p15.3–16 deletion were tumorigenic. Tumors were poorly differentiated ER‐α and progesterone receptor‐negative adenocarcinomas that expressed keratins, EMA, and E‐cadherin. Tumors and tumor‐derived cell lines exhibited loss of chromosome 4, deletions in chromosomes 3p12.3–13, 8p11.1–21, 9p21‐qter, and 18q, and gains in 1p, and 5q15‐qter. The induction of complete transformation of MCF‐10F cells in vitro confirms the carcinogenicity of E2, supporting the concept that this hormone could act as an initiator of breast cancer in women. This model provides a unique system for understanding the genomic changes that intervene for leading normal cells to tumorigenesis and for testing the functional role of specific genomic events taking place during neoplastic transformation.—Russo, J., Fernandez, S. V., Russo, P. A., Fernbaugh, R., Sheriff, F. S., Lareef, H. M., Garber, J., Russo, I. H. 17‐Beta‐estradiol induces transformation and tumorigenesis in human breast epithelial cells. FASEB J. 20, 1622–1634 (2006)

Estrogen and Xenoestrogens in Breast Cancer

There is growing concern that estrogenic environmental compounds that act as endocrine-disrupting chemicals might potentially have adverse effects on hormone-sensitive organs such as the breast. This concern is further fueled by evidence indicating that natural estrogens, specifically 17β-estradiol, are important factors in the initiation and progression of breast cancer. We have developed an in vitro—in vivo model in which we have demonstrated the carcinogenicity of E2 in human breast epithelial cells MCF-10F. Hypermethylation of NRG1, STXBP6, BMP6, CSS3, SPRY1, and SNIP were found at different progression stages in this model. The use of this powerful and unique model has provided a tool for exploring whether bisphenol A and butyl benzyl phthalate have relevance in the initiation of breast cancer. These studies provide firsthand evidence that the natural estrogen 17β-estradiol and xenoestrogenic substances like bisphenol A are able to induce neoplastic transformation in human breast epithelial cells.

Estradiol and its metabolites 4-hydroxyestradiol and 2-hydroxyestradiol induce mutations in human breast epithelial cells

An elevated incidence of breast cancer in women has been associated with prolonged exposure to high levels of estrogens. Our laboratory demonstrated that treatment of the immortalized human breast epithelial cells MCF‐10F with 17β‐estradiol (E2), 4‐hydroxyestradiol (4‐OHE2) or 2‐hydroxyestradiol (2‐OHE2) induces phenotypical changes indicative of neoplastic transformation. MCF‐10F cells treated with E2, 4‐OHE2 or 2‐OHE2 formed colonies in agar methocel and lost their ductulogenic capacity in collagen, expressing phenotypes similar to those induced by the carcinogen benzo[a]pyrene. To investigate whether the transformation phenotypes were associated with genomic changes, cells treated with E2, 4‐OHE2 or 2‐OHE2 at different doses were analyzed using microsatellite markers. Since microsatellite instability (MSI) and loss of heterozygosity (LOH) in chromosomes 13 and 17 have been reported in human breast carcinomas, we tested these parameters in MCF‐10F cells treated with E2, 2‐OHE2, or 4‐OHE2 alone or in combination with the antiestrogen ICI182780. MCF‐10F cells treated with E2 or 4‐OHE2, either alone or in combination with ICI182780, exhibited LOH in the region 13q12.3 with the marker D13S893 located at ∼0.8 cM telomeric to BRCA2. Cells treated with E2 or 4‐OHE2 at doses of 0.007 and 70 nM and 2‐OHE2 only at a higher dose (3.6 μM) showed a complete loss of 1 allele with D13S893. For chromosome 17, differences were found using the marker TP53‐Dint located in exon 4 of p53. Cells treated with E2 or 4‐OHE2 at doses of 0.007 nM and 70 nM and 2‐OHE2 only at a higher dose (3.6 μM) exhibited a 5 bp deletion in p53 exon 4. Our results show that E2 and its catechol estrogen metabolites are mutagenic in human breast epithelial cells. ICI182780 did not prevent these mutations, indicating that the carcinogenic effect of E2 is mainly through its reactive metabolites 4‐OHE2 and 2‐OHE2, with 4‐OHE2 and E2 being mutagenic at lower doses than 2‐OHE2.

Sodium/potasium ATPase (Na+, K+-ATPase) and ouabain/related cardiac glycosides : a new paradigm for development of anti- breast cancer drugs?

SummaryProlonged exposure to 17β-estradiol (E2) is a key etiological factor for human breast cancer. The biological effects and carcinogenic effects of E2 are mediated via estrogen receptors (ERs), ERα and ERβ. Anti-estrogens, e.g. tamoxifen, and aromatase inhibitors have been used to treat ER-positive breast cancer. While anti-estrogen therapy is initially successful, a major problem is that most tumors develop resistance and the disease ultimately progresses, pointing to the need of developing alternative drugs targeting to other critical targets in breast cancer cells. We have identified that Na+, K+-ATPase, a plasma membrane ion pump, has unique/valuable properties that could be used as a potentially important target for breast cancer treatment: (a) it is a key player of cell adhesion and is involved in cancer progression; (b) it serves as a versatile signal transducer and is a target for a number of hormones including estrogens and (d) its aberrant expression and activity are implicated in the development and progression of breast cancer. There are several lines of evidence indicating that ouabain and related digitalis (the potent inhibitors of Na+, K+-ATPase) possess potent anti-breast cancer activity. While it is not clear how the suggested anti-cancer activity of these drugs work, several observations point to ouabain and digitalis as being potential ER antagonists. We critically reviewed many lines of evidence and postulated a novel concept that Na+, K+-ATPase in combination with ERs could be important targets of anti-breast cancer drugs. Modulators, e.g. ouabain and related digitalis could be useful to develop valuable anti-breast cancer drugs as both Na+, K+-ATPase inhibitors and ER antagonists.

TP53 mutations detected in circulating tumor cells present in the blood of metastatic triple negative breast cancer patients

IntroductionCirculating tumor cells (CTCs) are tumor cells shed from either primary tumors or its metastases that circulate in the peripheral blood of patients with metastatic cancers. The molecular characterization of the CTCs is critical to identifying the key drivers of cancer metastasis and devising therapeutic approaches. However, the molecular characterization of CTCs is difficult to achieve because their isolation is a major technological challenge.MethodsCTCs from two triple negative breast cancer patients were enriched using CellSearch and single cells selected by DEPArray™. A TP53 R110 fs*13 mutation identified by next generation sequencing in the breast and chest skin biopsies of both patients was studied in single CTCs.ResultsFrom 6 single CTC isolated from one patient, 1 CTC had TP53 R110 delC, 1 CTC showed the TP53 R110 delG mutation, and the remaining 4 single CTCs showed the wild type p53 sequence; a pool of 14 CTCs isolated from the same patient also showed TP53 R110 delC mutation. In the tumor breast tissue of this patient, only the TP53 R110 delG mutation was detected. In the second patient a TP53 R110 delC mutation was detected in the chest wall skin biopsy; from the peripheral blood of this patient, 5 single CTC and 6 clusters of 2 to 6 CTCs were isolated; 3 of the 5 single CTCs showed the TP53 R110 delC mutation and 2 CTCs showed the wild type TP53 allele; from the clusters, 5 showed the TP53 R110 delC mutation, and 1 cluster the wild type TP53 allele. Single white blood cells isolated as controls from both patients only showed the wild type TP53 allele.ConclusionsWe are able to isolate uncontaminated CTCs and achieve single cell molecular analysis. Our studies showed the presence of different CTC sub-clones in patients with metastatic breast cancer. Some CTCs had the same TP53 mutation as their matching tumor samples although others showed either a different TP53 mutation or the wild type allele. Our results indicate that CTCs could represent a non-invasive source of cancer cells from which to determine genetic markers of the disease progression and potential therapeutic targets.

Epithelial to Mesenchymal Transition in Human Breast Epithelial Cells Transformed by 17β-Estradiol

The estrogen dependence of breast cancer has long been recognized; however, the role of 17β-estradiol (E2) in cancer initiation was not known until we showed that it induces complete neoplastic transformation of the human breast epithelial cells MCF-10F. E2 treatment of MCF-10F cells progressively induced high colony efficiency and loss of ductulogenesis in early transformed (trMCF) cells and invasiveness in Matrigel invasion chambers. The cells that crossed the chamber membrane were collected and identified as bsMCF; their subclones were designated bcMCF; and the cells harvested from carcinoma formation in severe combined immunodeficient mice were designated caMCF. These phenotypes correlated with gene dysregulation during the progression of the transformation. The highest number of dysregulated genes was observed in caMCF, being slightly lower in bcMCF, and lowest in trMCF. This order was consistent with the extent of chromosome aberrations (caMCF > bcMCF >>> trMCF). Chromosomal amplifications were found in 1p36.12-pter, 5q21.1-qter, and 13q21.31-qter. Losses of the complete chromosome 4 and 8p11.21-23.1 were found only in tumorigenic cells. In tumor-derived cell lines, additional losses were found in 3p12.1-14.1, 9p22.1-pter, and 18q11.21-qter. Functional profiling of dysregulated genes revealed progressive changes in the integrin signaling pathway, inhibition of apoptosis, acquisition of tumorigenic cell surface markers, and epithelial-mesenchymal transition. In tumorigenic cells, the levels of E-cadherin, epithelial membrane antigen, and various keratins were low and CD44E/CD24 were negative, whereas SNAI2, vimentin, S100A4, FN1, HRAS, transforming growth factor β1, and CD44H were high. The phenotypic and genomic changes triggered by estrogen exposure that lead normal cells to tumorigenesis confirm the role of this steroid hormone in cancer initiation. [Cancer Res 2007;67(23):11147–57]

Expression and DNA methylation changes in human breast epithelial cells after bisphenol A exposure

It has been suggested that xenoestrogens, a group of agents termed endocrine disruptors, may contribute to the development of hormone-dependent cancers, such as breast and endometrial cancers. We previously demonstrated that the xenoestrogen, bisphenol A (BPA), was able to induce the transformation in vitro of human breast epithelial cells. The normal-like human breast epithelial cell line, MCF-10F, formed tubules in collagen (3-D cultures), although after treatment with BPA (10-5 M and 10-6 M BPA) the cells produced less tubules (73% and 80%, respectively) and some spherical masses (27% and 20%, respectively). In the present study, expression and DNA methylation analyses were performed in these cells after exposure to BPA. These cells showed an increased expression of BRCA1, BRCA2, BARD1, CtIP, RAD51 and BRCC3, all of which are genes involved in DNA repair, as well as the downregulation of PDCD5 and BCL2L11 (BIM), both of which are involved in apoptosis. Furthermore, DNA methylation analysis showed that the BPA exposure induced the hypermethylation of BCL2L11, PARD6G, FOXP1 and SFRS11, as well as the hypomethylation of NUP98 and CtIP (RBBP8). Our results indicate that normal human breast epithelial cells exposed to BPA have increased expressions of genes involved in DNA repair in order to overcome the DNA damage induced by this chemical. These results suggest that the breast tissue of women with BRCA1 or BRCA2 mutations could be more susceptible to the effects of BPA.

Inflammatory breast cancer (IBC): clues for targeted therapies

Inflammatory breast cancer (IBC) is the most aggressive type of advanced breast cancer characterized by rapid proliferation, early metastatic development and poor prognosis. Since there are few preclinical models of IBC, there is a general lack of understanding of the complexity of the disease. Recently, we have developed a new model of IBC derived from the pleural effusion of a woman with metastatic secondary IBC. FC-IBC02 cells are triple negative and form clusters (mammospheres) in suspension that are strongly positive for E-cadherin, β-catenin and TSPAN24, all adhesion molecules that play an important role in cell migration and invasion. FC-IBC02 cells expressed stem cell markers and some, but not all of the characteristics of cells undergoing epithelial mesenchymal transition (EMT). Breast tumor FC-IBC02 xenografts developed quickly in SCID mice with the presence of tumor emboli and the development of lymph node and lung metastases. Remarkably, FC-IBC02 cells were able to produce brain metastasis in mice on intracardiac or intraperitoneal injections. Genomic studies of FC-IBC02 and other IBC cell lines showed that IBC cells had important amplification of 8q24 where MYC, ATAD2 and the focal adhesion kinase FAK1 are located. MYC and ATAD2 showed between 2.5 and 7 copies in IBC cells. FAK1, which plays important roles in anoikis resistance and tumor metastasis, showed 6–4 copies in IBC cells. Also, CD44 was amplified in triple-negative IBC cells (10–3 copies). Additionally, FC-IBC02 showed amplification of ALK and NOTCH3. These results indicate that MYC, ATAD2, CD44, NOTCH3, ALK and/or FAK1 may be used as potential targeted therapies against IBC.

Formation of depurinating N3Adenine and N7Guanine adducts by MCF-10F cells cultured in the presence of 4-hydroxyestradiol

Metabolic conversion of endogenous estrogens, estradiol (E2) and estrone (E1), to the catechol estrogens 4‐hydroxyE1(E2) [4‐OHE1(E2)] has been implicated in the initiation of cancer in rodents and humans. Evidence collected in our laboratories has shown that 4‐OHE1(E2) are enzymatically oxidized to E1(E2)‐3,4‐quinones [E1(E2)‐3,4‐Q], which have the potential to damage DNA by forming predominantly depurinating adducts, 4‐OHE1(E2)‐1‐N3Ade and 4‐OHE1(E2)‐1‐N7Gua, leading to the accumulation of mutations and probably cell transformation. The human breast epithelial cell line MCF‐10F has been transformed by treatment with E2 or 4‐OHE2. We have used MCF‐10F cells to study the presence of adducts and conjugates after treatment with 4‐OHE2. To mimic the intermittent exposure of breast cells to endogenous estrogens, MCF‐10F cells were treated with 1 μM 4‐OHE2 for a 24‐h period at 72, 120, 192 and 240 h postplating. Culture media were collected at each point, extracted by solid‐phase extraction and analyzed by HPLC connected with a multichannel electrochemical detector and/or ultraperformance liquid chromatography/tandem mass spectrometry. Media from successive treatments with 4‐OHE2 showed the formation of methoxy and cysteine conjugates, and the depurinating adducts 4‐OHE1(E2)‐1‐N3Ade. The amount of 4‐OHE1(E2)‐1‐N3Ade adducts was higher after the third treatment; smaller amounts of the 4‐OHE1(E2)‐1‐N7Gua adducts were detected after the second and third treatments. These results demonstrate that MCF‐10F cells oxidize 4‐OHE2 to E1(E2)‐3,4‐Q, which react with DNA to form the depurinating N3Ade and N7Gua adducts. This DNA damage can play an important role in the 4‐OHE2‐induced mutations and transformation of MCF‐10F cells to malignant cells.

Human chorionic gonadotropin (hCG) prevents the transformed phenotypes induced by 17 β-estradiol in human breast epithelial cells.

Human chorionic gonadotropin (hCG), a hormone produced during pregnancy, can elicit life‐long refractoriness to carcinogenesis by differentiation of the breast epithelium. Human breast epithelial cells MCF‐10F form tubules in collagen, mimicking the normal ductules. We have shown that 17 β‐estradiol (E2) alter the ductulogenic pattern of these cells. The effect of the recombinant hCG (rhCG) in vitro was evaluated on the transformation of MCF‐10F induced by E2. MCF‐10F cells were treated with 70 nM E2 alone or in combination with 50 IU/ml rhCG during 2 weeks, while the controls were treated with DMSO (the solvent in which E2 was dissolved) or rhCG alone. At the end of treatment, the cells were plated in type I collagen matrix (3D‐cultures) for detecting 2 main phenotypes of cell transformation, namely the loss of ductulogenic capacity and the formation of solid masses. Although E2 significantly increased solid mass formation, this effect was prevented when MCF‐10F cells were treated with E2 in combination with rhCG. Furthermore, E2 increased the main duct width (p < 0.001), and caused a disruption of the luminal architecture, whereas rhCG increased the length of the tubules (p < 0.001) and produced tertiary branching. In conclusion, rhCG was able to abrogate the transforming abilities of estradiol, and had the differentiating property by increasing the branching of the tubules formed by breast epithelial cells in collagen. These results further support our hypothesis, known as the terminal differentiation hypothesis of breast cancer prevention, that predicts that hCG treatment results in protection from tumorigenic changes by the loss of susceptible stem cells 1 through a differentiation to refractory stem cells 2 and increase differentiation of the mammary gland.