The major goal of this project is to determine whether estrogens induce neoplastic transformation of the normal human breast epithelial cells MCF-10F through non-receptor-mediated hormonal activities.
This research project was initiated by the NCI in 1996 as a Complementary Collaborative Coalition of researchers interested in the role of estrogen in cancer. From this initial group a consortium was formed by FCCC, University of Nebraska, University of Memphis, University of Virginia, and New York University School of Dentistry which developed into the Breast Cancer Center of Excellence, funded by the Department of Defense. Collaboration with Dr. Maria Luiza Mello and Dr. Benedicto Vidal from the University of Campinas, Brazil has generated information on the changes in nuclear structure of estrogen induced cell transformation. The study on the epithelial-mesenchymal transition of breast epithelial cells transformed by estrogen was performed in collaboration with Dr. Thomas Sutter from the University of Memphis in collaboration with Dr. Daniel Tiezzi (University of Ribeiro Preito, Brazil) in the epithelial mesenchymal transition of breast epithelial cells transformed by estrogen has been part of Dr. Tiezzi’s doctoral thesis. Altogether, this project has generated novel insights in the molecular basis of estrogen-induced carcinogenesis.
Breast cancer is a malignancy whose dependence on estrogen exposure has long been recognized, even though the mechanisms through which estrogens cause cancer are not clearly understood. This work was performed in order to determine whether 17ß-estradiol (E2), the predominant circulating ovarian steroid, is carcinogenic in human breast epithelial cells and whether non-receptor mechanisms are involved in the initiation of breast cancer. For this purpose, the effect of four alternating 24 hr treatment periods with 70nM E2 of the estrogen receptor alpha (ER-α) negative MCF-10F cell line on the in vitro expression of neoplastic transformation was evaluated (Figure 21: Click to see).
E2-treatment induced the expression of anchorage independent growth, loss of ductulogenesis in collagen, invasiveness in Matrigel, and loss of 9p11-13. Tumorigenesis in SCID mice was expressed only in invasive cells that in addition exhibited a deletion of 4p15.3-16. Tumors formed in SCID mice were poorly differentiated adenocarcinomas that were estrogen receptor α and progesterone receptor negative, expressed keratins, EMA and e-cadherin (Figure 22: Click to see).
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, leading normal cells to tumorigenesis, and for testing the functional role of specific genomic events taking place during neoplastic transformation.
Drs. Johana Vanegas and Patricia Russo in the BCRL performed time lapse photography in order to study the motility of MCF 10F and the effect of estrogens and its metabolites on cell motility and migratory movements.
The relationship between cell motility in vitro and the ability of neoplastic cells to invade and metastasize in vivo is well known. Thisled us to evaluate the migratory behavior of the human breast epithelial cell line MCF-10F after neoplastic transformation with 70nM of 17ß-estradiol (E2), 4 OH estradiol (4-OH-E2) and 2-OH estradiol (2-OH-E2). Cells thus transformed express colony formation in agar methocel, loss of ductulogenic capacity in collagen matrix, and invasiveness in a Matrigel artificial membrane. Time-lapse video microscopy was used to directly observe and capture the cells’ images using a Nikon DXM digital camera attached to an Olympus IMT-2 microscope that was equipped with a Plexiglas incubation chamber. Cells were maintained in 5% CO2 at 37°C with 95% humidity. The cells were photographed at 10x and 20x magnifications at 120 sec intervals for periods ranging from 24 to 72hrs. Metamorph® software version 6.1r6 was used to obtain images that were then compiled into “stacks” images played back at 1/30th of a second frame rate. From each cell line a random number of cells were selected for tracking at 1 hour-intervals. Cell motility was evaluated by determining the speed of the tracked cell expressed in mm/min (S), the direction persistence in time (P), and the random motility coefficient (µ) that provides a measure of how fast a cell population will grow to cover a surface. In E2 transformed MCF-10F cells the speed (S) and random motility (µ) were not significantly different from the same parameters measured in control cells, whose motility was: S=0.31±0.06, and µ=3.8±1.5, whereas cell direction persistence in time was P=126.9±39.1, which was significantly higher than in the control (P=78±13) (p<0.04). Cells transformed with 4-OH-E2 showed a significant increase in cell speed (S=0.44±0.14) (p<0.005), cell direction persistence in time (P=131±53) (p<0.05), and random motility coefficient (µ=15.3±12.4) (p<0.05). Those cells transformed by 2-OH-E2 showed an increase in S=0.47±0.22 (p<0.07), and P=120±51 (p<0.03), whereas the random motility coefficient was not significantly modified.). Our findings indicate that the transformation of HBEC by estrogen and its metabolites induce changes in cell motility in vitro, 4-OH-E2 being the one inducing the most significant changes, its effect correlated to the expression of phenotypes indicative of cell transformation (Russo J et al, J Steroid Biochem Mol Biol 87:1, 2003).
The estrogen-dependence of breast cancer has long been recognized, however, the role of 17ß-estradiol (E2) in cancer initiation was not known until we demonstrated 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, invasiveness in a Matrigel invasion chambers. The cells that crossed the chamber membrane were collected and identified as bsMCF, and their subclones designated bcMCF, and the cells harvested from carcinoma formation in SCID mice designated (caMCF) (Figure 23: Click to see).
These phenotypes correlated with gene dysregulation during the progression of the transformation. The highest number of dysregulated genes was observed in caMCF cells, being slightly lower in bcMCF cells, and lowest in trMCF cells. 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 of 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 to mesenchymal transition. In tumorigenic cells, the levels of E-cadherin, EMA, and various keratins were low and CD44E/CD24 were negative, whereas SNAI2, vimentin, S100A4, FN1, HRAS and TGFβ1, and CD44H were high (Figure 24: Click to see).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.