Estrogens as carcinogens in human breast
Authors: Jose Russo
Journal: Cancer Research (2005)

Proc Amer Assoc Cancer Res, Volume 46, 2005

SY14-1 

Even though the breast is influenced by a myriad of hormones and growth factors, estrogens are considered to play a major role in promoting the proliferation of both the normal and the neoplastic breast epithelium. Their role as breast carcinogens has long been suspected and recently confirmed by epidemiological studies. However, the ultimate mechanism through which estrogens transform a normal into a neoplastic cells has not been unraveled yet. Three major mechanisms are postulated to be involved in their carcinogenic effects: stimulation of cellular proliferation through their receptor-mediated hormonal activity, resulting in more opportunities for accumulation of genetic damages leading to carcinogenesis, direct genotoxic effects by increasing mutation rates through a cytochrome P450-mediated metabolic activation, and induction of aneuploidy. There is also evidence that estrogens compromise the DNA repair system, leading to the accumulation of genomic lesions that are essential to estrogen-induced carcinogenesis. To fully demonstrate that estrogens are carcinogenic in the human breast and for testing potential mechanisms of action it is required an experimental system in which the natural estrogen 17β-estradiol (E2) by itself or its metabolites 2-hydroxy, 4-hydroxy, and 16-a-hydroxy-estradiol (2-OH-E2,4-OH-E2, and 16-α-OH E2) respectively, would induce neoplastic transformation of human breast epithelial cells (HBEC) in vitro to a degree at least similar to that induced by the chemical carcinogen benz(a)pyrene (BP) ([1][1],[2][2]) The transforming potential of estrogens on the human breast epithelium was evaluated by treating the spontaneously immortalized estrogen receptor alpha (ERα) negative MCF-10F cells with 0.007nM, 70nM and 1μg/ml of E2, 2-OH-E2, 4-OH-E2, and 16-α-OH E2, or cholesterol ([1][1]). Treatments with estrogens alone or in the presence of the antiestrogens tamoxifen (TAM) or ICI-182,780 were done for 24 hours twice a week for two weeks in order to mimic the intermittent exposure of the breast to endogenous estrogens. . At the end of the second week of treatment, and in successive passages thereafter, the cells were evaluated for assessing the expression of phenotypes indicative of cell transformation ([1][1]–[3][3]), namely, determination of colony formation in agar-methocel, or colony efficiency (CE), ductulogenic capacity in collagen matrix, invasiveness in a reconstituted basement membrane using the Boyden chamber, genomic analysis by capillary electrophoresis, and tumorigenic assay in severe compromised immune-deficient mice (scid), which have the striking capability of discriminating between normal and neoplastic cells. At all passages tested MCF-10F cells treated with BP, E2, 2-OH-E2, 4-OH-E2, and 16α-OH-E2 formed colonies in agar methocel that were greater than 80 μm in diameter. Cells treated with cholesterol did not form colonies. CE was dose dependent and similar in cells treated with BP and E2; 2-OH-E2 treated cells had lower CE than the two previous compounds, and in 4-OH-E2-treated cells CE was greater at the dose of 0.007nM, reaching a plateau at the two higher doses. Ductulogenesis, which was evaluated by estimating the ability of cells plated in collagen to form tubules, revealed that cholesterol treated control MCF-10F cells formed ductule-like structures mimicking the normal branching pattern of the mammary parenchyma. The ductulogenic capacity was lost in BP and E2 treated cells, which formed instead solid masses. The metabolites of estrogen also impaired the formation of ductules. Histological analysis showed that the ductules that control cells formed in the collagen matrix were lined by a single layer of cuboidal epithelial cells. E2 the E2-metabolite-treated cells formed spherical masses filled by large cuboidal cells. The invasive capacity of MCF-10F cells was significantly increased by BP; E2, and 4OH-E2 treatments increased even further the cells’ invasiveness; 2-OH-E2 treatment stimulated invasiveness but to a lower degree than BP. Neither TAM nor ICI-182,780 abrogated the transforming efficiency of estrogen metabolites. Genomic analysis revealed that MCF-10F cells transformed with E2 and 4-OH-E2 either alone or in combination with ICI exhibited loss of heterozygosity (LOH) in the region 13q12.3 (D13S893 marker located at approximately 0.8cM telomeric to BRCA2) at all the doses tested; 2-OH-E2 induced the same change only at the highest dose used (1μg/ml). Another significant genomic change observed was a 5bp deletion in TP53 exon 4 of chromosome 17 (marker TP53-Dint located in exon 4 of TP53) in cells treated with E2 and 4-OH-E2 at the doses of 0.007nM and 70nM and with 2-OH-E2 only at a highest dose used. These changes were considerd to be specific for estradiol and its metabolites, since the observed mutations in HBEC at D13S893 and TP53 exon 4 loci were not induced by BP treatment. Injection of 10-15X106 control and treated cells in the inguinal fat pad of SCID mice failed to induce tumors up the 9th passage. In order to determine whether more aggressive phenotypes could be selected, cells in their 9th passage after transformation with E2 were seeded in a Boyden chambers, and those cells crossing the membrane were collected, expanded, and designated E2-70- B2,B3,B4,B5,C2,C3,C4 and C5 to those transformed with 70nM and 1-B2, 1-B3, 1-B4, 1-B5, 1-C2, 1-C3, 1-C4, qnd 1-C5 to those transformed with 1μM E2. These cells were injected to SCID mice for tumorigenic assay. Only E2-70-C3 and E2-70-C5 were tumorigenic in 2/12 and 9/10 animals injected, respectively. The tumors were poorly differentiated adenocarcinomas, Erα, Erβ and progesterone receptor negative. negative, expressing immunocytochemically high molecular weight basic keratin (+++), E Cadherin (+), CAM5.2 (+) and vimentin (+++). RNA was collected from E2-70-C3 and E2-70-C5 cells for cDNA microarray analysis. The genomic profile of E2-70-C3 differed from that of E2-70-C5 cells, since this latter one overexpressed by more than 5-fold genes such as tankyrase (TRF1-interacting ankyrin related ADP ribose polymerase), claudin 1, homeobox C10, and Notch homolog 3; it also exhibited downregulation of telomeric repeat binding factor and tumor metastasissuppressor gene, all genes that have been shown to be altered in primary breast cancer. From the 9 tumors obtained fro E2-70-C5 cells four tumoral cell lines designated C5-A1-T1,C5-A4-T4, C5-A6-T6 and C5-A8-T8 were derived. Fingerprint analysis confirmed that all these cells originated from MCF-10F cells. Comparative genomic hybridization (CGH) analysis of that E2-70-C5 derived tumors exhibited gains in chromosomes 1p, 5q15-qter and losses of chromosome 4, 3p12.3-13, 8p11.1-21, 9p21-pter and 18q. All together we have accumulated evidence indicating that E2 and and its metabolites are mutagenic as an early event in the process of transformation of the human breast epithelium. The fact that an antiestrogen did not prevent these mutations, indicated that the carcinogenic effect of this hormone and its metabolites is independent of the receptor pathway. Whereas these early mutational events were associated with the emergence of transformation phenotypes such as colony formation, loss of ductulogenic capacity, and invasiveness, they were not enough for inducing the tumorigenic phenotype, which seemed to require further selection and amplification of genes such as ankyrase, resulting in multiple losses in multiple putative tumor suppressor genes located on chromosomes 4, 3, 8 and 9, whose inactivation is important in the pathogenesis of breast cancer. (This work was supported by grants DMAD17-00-1-0247, DMAD17-03-1-0229).

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