Although the role of estrogen signaling in breast cancer development has been extensively studied, the mechanisms that regulate the indispensable role of estrogen in normal mammary gland development have not been well studied. Because of the unavailability of culture system to maintain estrogen-receptor-positive (ERα+) cells in vitro, the molecular mechanisms that regulate estrogen/ERα signaling in the normal human breast are unknown. In the present study, we examined the effects of estrogen signaling on ERα+ human luminal progenitors using a modified matrigel assay and found that estrogen signaling increased the expansion potential of these progenitors. Furthermore, we found that blocking ERα attenuated luminal progenitor expansion and decreased the luminal colony-forming potential of these progenitors. Additionally, blocking ERα decreased H19 expression in the luminal progenitors and led to the development of smaller luminal colonies. We further showed that knocking down the H19 gene in the luminal progenitors significantly decreased the colony-forming potential of the luminal progenitors, and this phenotype could not be rescued by the addition of estrogen. Lastly, we explored the clinical relevance of the estrogen–H19 signaling axis in breast tumors and found that ERα+ tumors exhibited a higher expression of H19 as compared with ERα− tumors and that H19 expression showed a positive correlation with ERα expression in those tumors. Taken together, the present results indicate that the estrogen–ERα–H19 signaling axis plays a role in regulating the proliferation and differentiation potentials of the normal luminal progenitors and that this signaling network may also be important in the development of ER+ breast cancer tumors.
Pratima Basak, Sumanta Chatterjee, Steven Weger, M Christine Bruce, Leigh C Murphy, and Afshin Raouf
Mathieu Lupien and Myles Brown
Alterations in transcription programs are a fundamental feature of cancer. Nuclear receptors, such as the estrogen receptor alpha (ERα) and androgen receptors (ARs), are central in this process as they can directly impact gene expression through interaction with the chromatin and subsequent association with coregulators and the transcriptional machinery. Unbiased genome-wide investigations have demonstrated the predominant recruitment of both ERα and AR to distant (non-promoter)-regulatory elements. Furthermore, these studies revealed a clear relationship between sites of transcription factor recruitment and gene regulation. Indeed, expression profiles from AR-positive primary prostate tumors and cell lines directly relate to the AR cistrome in prostate cancer cells, while the ERα cistrome in breast cancer cells relates to expression profiles from ERα-positive primary breast tumors. Additionally, cell-type-specific ERα cistromes are linked to lineage-specific estrogen-induced expression profiles in different cell types, for example osteosarcoma and breast cancer cells. The pioneer factor forkhead box A1 (FoxA1/HNF3α) plays a central role in AR and ERα signaling. It is recruited in a lineage-specific manner translating the epigenetic signature consisting of mono- and dimethylated histone H3 on lysine 4 (H3K4me1/me2) into functional regulatory elements. Hence, through the interplay between the pioneer factor, namely FoxA1, and epigenetic events, the transcriptional potential of a given cell lineage is predefined. Since this directly impacts signaling through nuclear receptors, these discoveries should significantly impact the development of novel therapeutic strategies directed against multiple types of cancer.
Vanessa W Lim, Jun Li, Yinhan Gong, Aizhen Jin, Jian-Min Yuan, Eu Leong Yong, and Woon-Puay Koh
The estrogen levels of Asian women are different from those of Western women, and this could affect estrogen receptor (ER) bioactivity and breast cancer risk. We conducted a case–control study in 169 postmenopausal breast cancer cases and 426 matched controls nested within a population-based prospective cohort study, the Singapore Chinese Health Study, to evaluate the serum levels of estrogens and their receptor (ERα and ERβ)-mediated estrogenic activities in relation to breast cancer risk. Breast cancer cases had higher levels of estrogens and ER-mediated bioactivities in baseline serum than the controls. Compared with those in the lowest quartile, women in the highest quartile for estrone (E1) or ERα-mediated bioactivity had increased breast cancer risk. After additional adjustment for ERβ bioactivity, free estradiol, and E1 levels, serum ERα-mediated bioactivity remained associated with increased breast cancer risk. Compared with those in the lowest quartile, women in the highest quartile for ERα-mediated bioactivity had an odds ratio of 2.39 (95% CI=1.17–4.88; P for trend=0.016). Conversely, the positive association between E1 and cancer risk became null after adjustment for ERα-mediated bioactivity, suggesting that the effect of E1 could be mediated through ERα. Factor(s) contributing to increased ERα-mediated estrogenic bioactivity in serum and its role as a predictor for breast cancer risk need to be validated in future studies.
E Levin, S Caruso, and A M Actis
L E Yaich, N Roodi, L R Bailey, C S Verrier, C J Yee, D R Cavener, and F F Parl
The estrogen receptor (ER) content of breast cancers predicts the likelihood of benefit from antiestrogen therapy. Tumors with an ER content of less than 10 fmol/mg are considered to be ER-negative and have a poor response rate to tamoxifen. The molecular basis of the ER-negative phenotype is not well understood. This study was conducted in five ER-positive (MCF-7, T47D, ZR-75-1, MDAMB-134, MDA-MB-361) and five ER-negative (BT-20, HBL-100, MDA-MB-157, MDA-MB-231, MDA-MB-468) human breast cancer cell lines to determine whether the ER-negative phenotype results from mutations in the coding region of the ER gene or is due to a deficiency of the transcriptional or post-transcriptional regulation of ER expression. The coding region was examined for mutations by denaturing gradient gel electrophoresis, single-strand conformation polymorphism and DNA sequence analysis. The presence and integrity of ER mRNA were investigated by Northern blotting and reverse transcription (RT)-PCR amplification. The presence and concentration of ER protein were assessed by Western blotting and hormone-binding assay. Both ER-negative and -positive cell lines contained silent mutations in codons 10, 325 and 594 of the ER gene, all of which probably represent neutral polymorphic sites. No missense or nonsense mutations were identified in any of the cell lines. About 2 kb of the 5′ upstream region and 4.3 kb of the 3′ untranslated region of the ER gene were present and grossly intact in all cell lines. By Northern blotting, ER mRNA was shown to be present in all ER-positive cell lines and, with the exception of BT-20 cells, absent in all ER-negative cell lines. However, ER mRNA was found to be detectable in all ER-negative cell lines when using the more sensitive RT-PCR. ER protein was undetectable by Western blotting in all cell lines that were ER-negative by ligand-binding assay. This study indicates that the ER-negative phenotype of breast cancer cell lines is not the result of ER gene mutations. It is due to the actual absence of ER protein which results from deficient ER expression at the transcriptional or post-transcriptional level.
Endocrine-Related Cancer (1995) 2 293-309
R Clarke and N Brünner
Almost all initially responsive breast tumors acquire resistance to the triphenylethylene antiestrogen tamoxifen (TAM). Development of resistance represents the major restriction in the current use of TAM. More recently, a series of steroidal antiestrogens have been developed (e.g. ICI 182,780), and we wished to determine whether cross-resistance among these drugs occurs, and what the likely mechanisms of resistance are. We have further selected breast cancer variant cell lines that no longer require estrogens for growth (MCF-7/LCC-1) against either 4-hydroxytamoxifen (MCF-7/LCC-2) or ICI 182,780 (MCF-7/LCC-9). Analysis of the resistance phenotypes suggests that TAM resistant cells can retain responsiveness to the steroidal antiestrogens, whereas cells selected against ICI 182,780 are crossresistant to TAM. If similar resistance patterns are acquired in patients, a sequential modality with TAM as the first-line and ICI 182,780 as a second-line drug, is suggested. We have generated a gene-network hypothesis to explain these differential resistance patterns and outline their potential molecular mechanisms.
Endocrine-Related Cancer (1995) 2 59-72
Ton van Agthoven, Anieta M Sieuwerts, Danielle Meijer, Marion E Meijer-van Gelder, Thecla L A van Agthoven, Roya Sarwari, Stefan Sleijfer, John A Foekens, and Lambert C J Dorssers
Although endocrine treatment of breast cancer is effective and common practice, in advanced disease the development of resistance is nearly inevitable. To get more insight into individual genes that account for resistance against hormonal agents, we have executed functional genetic screens and subsequently evaluated the clinical relevance of several identified genes with respect to tumor aggressiveness and tamoxifen resistance in estrogen receptor-positive patients. Estrogen-dependent human breast cancer cells were transduced with different retroviral cDNA expression libraries and subjected to selective cultures with various anti-estrogens. From a total of 264 resistant cell clones, 132 different genes were recovered by PCR. By applying stringent selection criteria, we identified 15 breast cancer anti-estrogen resistance (BCAR) genes individually yielding resistance. BCAR genes were recovered with differential frequencies for the diverse culture conditions and anti-estrogen drugs. Analysis of the relation of BCAR genes (EIF1, FBXL10, HRAS, NRG1, PDGFRA, PDGFRB, RAD21, and RAF1) with tamoxifen treatment in patients with advanced disease showed significant association with clinical benefit and progression-free survival for EIF1 and PDGFRA mRNA levels. Furthermore, PDGFRA and HRAS mRNA levels were significantly associated with tumor aggressiveness in lymph node-negative patients who had not received adjuvant systemic therapy. In conclusion, our functional genetic screens showed that BCAR genes differ in their ability to confer resistance towards distinct anti-estrogens. Based on the clinical relevance of several BCAR genes, further studies are warranted to characterize the underlying mechanisms, which may ultimately lead to the development of novel treatments and more individualized management of breast cancer patients.
Susanna Vuorenoja, Bidut Prava Mohanty, Johanna Arola, Ilpo Huhtaniemi, Jorma Toppari, and Nafis A Rahman
Lytic peptide Hecate (23-amino acid (AA)) fused with a 15-AA fragment of human chorionic gonadotropin-β (CG-β), Hecate-CGβ conjugate (H-CGβ-c) selectively binds to and destroys tumor cells expressing LH/chorionic gonadotropin receptor (Lhcgr). Transgenic mice (6.5 month old) expressing SV40 T-antigen under the inhibin-α promoter (inhα/Tag) presenting with Lhcgr expressing adrenal tumors were treated either with H-CGβ-c, GnRH antagonist (GnRH-a), estradiol (E2; only females) or their combinations for 1 month. We expected that GnRH-a or E2 in combination with H-CGβ-c could improve the treatment efficacy especially in females by decreasing circulating LH and eliminating the potential competition of serum LH with the H-CGβ-c. GnRH-a and H-CGβ-c treatments were successful in males (adrenal weights 14±2.8 mg and 60±26 vs 237±59 mg in controls; P<0.05). Histopathologically, GnRH-a apparently destroyed the adrenal parenchyma leaving only the fibrotic capsule with few necrotic foci. In females, H-CGβ-c was totally ineffective, whereas GnRH-a (19±5 mg) or E2 (77±50 mg) significantly reduced the adrenal weights compared with controls (330±70 mg). Adrenal morphometry, cell proliferation markers, post-treatment suppression of serum progesterone, and quantitative RT-PCR of GATA-4, Lhcgr, and GATA-6 further supported the positive outcome. H-CGβ-c selectively killed the Lhcgr expressing tumor cells, whereas GnRH-a blocked tumor progression through gonadotropin suppression, emphasizing the gonadotropin dependency of these adrenocortical tumors. If extrapolated to humans, H-CGβ-c could be considered for the treatment of gonadotropin-dependent adrenal tumors in males, whereas in females gonadotropin suppression, but not H-CGβ-c, would work better.
R J Santen, W Yue, F Naftolin, G Mor, and L Berstein
Substantial evidence supports the concept that estrogens cause breast cancer in animals and in women but the precise mechanism is unknown. The most commonly held theory is that estrogens stimulate proliferation of breast cells and thus statistically increase the chances for genetic mutations which could result in cancer. Another theory is that estrogen metabolism generates oxygen-free radicals and quinones which produce both stable and unstable DNA adducts. Both result in genetic mutations which accumulate and could ultimately cause cancer. A major criticism of the latter hypothesis is that breast tissue contains insufficient concentrations of estrogen for accumulation of genotoxic metabolites. Our hypothesis is that breast tissue estrogen levels, as a result of in situ synthesis, are much higher than previously thought. We and others have shown that estrogen can be made in the breast itself through conversion of androgens to estrogens, a process catalyzed by the enzyme aromatase. The levels of estrogen in the breast increase when aromatase is overexpressed. With sufficient amounts of aromatase in breast tissue, enough estradiol as substrate should be available to allow formation of substantial amounts of genotoxic metabolites. We postulate that aromatase overexpression may in this way cause breast cancer. As evidence supporting this concept, four animal models of aromatase overexpression and either breast cancer or premalignant lesions have been described. We have provided evidence that normal breast tissue can make estrogen and that certain stimulatory compounds can increase aromatase activity in the breast by nearly 10,000-fold. If our concepts are correct, it might be possible to prevent breast cancer by blocking the aromatase enzyme. Drugs are currently available to inhibit aromatase nearly completely without causing significant side-effects. Aromatase inhibitors might be more effective than antiestrogens in preventing breast cancer because of their dual role to block both initiation and promotion of breast cancer. To inhibit the initiation process, these inhibitors would reduce levels of the genotoxic metabolites of estradiol by lowering estradiol concentrations in tissue. At the same time, aromatase inhibitors would inhibit the process of tumor promotion by lowering tissue levels of estradiol and thus blocking cell proliferation. These concepts provide a strong rationale for studies of aromatase inhibitors to prevent breast cancer.
P de Cremoux, C Tran-Perennou, B L Brockdorff, E Boudou, N Br√ºnner, H Magdel√©nat, and A E Lykkesfeldt
Using a quantitative real-time RT-PCR technique we have compared the expression of a number of genes in two different human breast cancer model systems for development of acquired resistance to antiestrogens. The model system developed at the Danish Cancer Society comprises the cell lines MCF-7, MCF-7/TAMR-1, MCF-7/182R-6 and MCF-7/182R-7, and the model system developed at the Lombardi Cancer Research Center consists of the cell lines MCF-7/LCC1, MCF-7/LCC2 and MCF-7/LCC9. The findings on the well-known parameters estrogen receptor (ER)alpha, progesterone receptor (PR) and epidermal growth factor receptor (EGFR) are in good agreement with previous reports, thus documenting the usefulness of the real-time RT-PCR technique for multiparametric RNA analysis. The gene expression levels in the two model systems were found to be quite similar in relation to ERalpha, AIB1 (amplified in breast cancer-1), breast cancer antiestrogen resistance gene 1 (BCAR1) and ErbB-2 mRNA expression, whereas significant differences were observed on the expression of ERbeta, multidrug resistance gene 1 (MDR1), PR and EGFR. Furthermore, the presented data suggest that ERbeta, AIB1, BCAR1, CYP19 and MDR1 are unlikely to be causally involved in development of antiestrogen resistance in these breast cancer cell lines.