Overexpression of insulin-like growth factor II (IGFII) in ZR-75-1 human breast cancer cells: higher threshold levels of receptor (IGFIR) are required for a proliferative response than for effects on specific gene expression

Previous transfection experiments using a zinc-inducible expression vector have shown that overexpression of insulin-like growth factor II (IGFII) in MCF7 human breast cancer cells can reduce dependence on oestrogen for cell growth in vitro (DALY RJ, HARRIS WH, WANG DY, DARBRE PD. (1991) Cell Growth Differentiation 2, 457-464.). Parallel transfections now performed into another oestrogen-dependent human breast cancer cell line (ZR-75-1) yielded three clones of transfected ZR-75-1 cells that produced levels of zinc-inducible IGFII mRNA and secreted mature IGFII protein similar to those found in the transfected MCF7 cells. However, unlike in MCF7 cells, no resulting effects were found on cell growth in the ZR-75-1 clones, even though the ZR-75-1 clones possessed receptors capable of binding 125I-IGFI and showed a growth response to exogenously added IGFII. Medium conditioned by the ZR-75-1 clones could stimulate growth of untransfected MCF7 cells, indicating that the secreted IGFII protein was bioactive. Furthermore, zinc-induced IGFII was capable of increasing both pS2 mRNA levels and CAT activity from a transiently transfected AP1-CAT gene in the ZR-75-1 clones. Constitutive co-overexpression of the protein processing enzyme PC2 resulted in reduced levels of large forms of zinc-inducible IGFII, but zinc treatment still produced no effect on cell growth rate. Finally, however, constitutive co-overexpression of the type I IGF receptor (IGFIR) did result in zinc-inducible increased basal cell growth and reduced dependence on oestrogen for cell growth. These results demonstrate that while overexpression of IGFII per se was sufficient to deregulate MCF7 cell growth, the ZR-75-1 cells are limited in their proliferative response by their intrinsic receptor levels. However, although the proliferative response was limited, molecular responses (expression of pS2 and AP1-CAT) were not limited, indicating that different cellular responses can have different threshold receptor level requirements.     

Are breast tumours resistant to tamoxifen also resistant to pure antioestrogens?

A substantial proportion of patients with breast cancer are treated with the antioestrogen tamoxifen. As with other endocrine therapies, clinical experience has shown that some tumours in which growth is initially attenuated by tamoxifen treatment become resistant to continued drug treatment and resume growth. The mechanisms underlying the development of tamoxifen resistance have yet to be described but represent an important focus of research with the aim of defining what other therapies might be effective following tamoxifen treatment. Secondly, an understanding of tamoxifen resistance might suggest means to develop more effective agents for primary treatment of the disease. The development of pure antioestrogens, for example ICI 164,384 and ICI 182,780, which differ pharmacologically from tamoxifen in being entirely free of oestrogen partial-agonist activity, together with cell and animal models of tamoxifen resistant human breast cancer, has revealed one mechanism which might be of considerable clinical significance. Pure antioestrogens were shown to inhibit the proliferation of a greater proportion of tumor cells than tamoxifen in vitro, a differential effect that was attributed to the oestrogenic activity of tamoxifen. Subsequently, cell culture studies have shown that breast cancer cell lines selected for resistance to tamoxifen can still remain sensitive to the growth inhibitory action of pure antioestrogens. Similarly, the growth of human breast tumours in nude mice, which is initially attenuated by tamoxifen but then resumes, can be inhibited by pure antioestrogens. Both types of experiment are consistent with the view that tamoxifen resistance in these model systems is due to the oestrogenic action of tamoxifen. Thus, it can be predicted that in some patients whose tumours recur during tamoxifen therapy, a further response to pure antioestrogen treatment might occur. Studies to examine this hypothesis are currently being undertaken with ICI 182,780. One mechanism which might account for the experimental observations is an intrinsic heterogeneity amongst breast tumour cells in their response to tamoxifen, i.e. that there are at least two different populations of cells; one population which responds to tamoxifen as an antioestrogen and one which “reads” tamoxifen as an oestrogen. The growth advantage thus conferred on the latter population would lead to its predominance. If this is what actually happens in a proportion of human tumours, it can be argued that primary treatment of the tumour with a pure antioestrogen, rather than tamoxifen, would be preferred since a more complete and longer-lasting response would be predicted. Recent comparative studies with human breast tumours grown in nude mice support these predictions.     

Expression of estrogen receptor alpha and beta in myometrium of premenopausal and postmenopausal women

Although a clear role for estrogen receptor (ER) alpha has been established, the contribution of ERbeta in estrogen-dependent development, growth and functions of the myometrium is not understood. As a first step towards understanding the role of ERbeta, we have examined the expression of ERalpha and ERbeta in the human myometrium. With competitive RT-PCR assays, the level of ERbeta mRNA was 10-200 times lower than that of ERalpha mRNA in both premenopausal and postmenopausal myometrium. In premenopausal myometrium, the expression pattern of ERbeta mRNA during the menstrual cycle was similar to that of ERalpha mRNA, with highest levels in peri-ovulatory phase. In postmenopausal myometrium, ERbeta mRNA was significantly higher than it was in premenopausal myometrium, while the level of ERalpha mRNA was lower. The net result was a change in the ratio of ERbeta to ERalpha mRNA expression. The ratio changed from 0.6-1.5 in premenopausal to 2.5-7.6 in postmenopausal myometrium. In premenopausal women, the gonadotropin releasing hormone analogue, leuprorelin acetate, elicited a decrease in ERalpha and an increase in ERbeta mRNA expression to cause a postmenopausal receptor phenotype. Estradiol, on the other hand, reversed ERalpha and ERbeta mRNA expression and their ratio in postmenopausal myometrium to those of premenopausal myometrium. Immunohistochemical staining and Western blot analysis of ERalpha and ERbeta with semiquantitative analysis showed good agreement between mRNA and protein levels. The data indicate that coordinated expression of ERalpha and ERbeta might be necessary for normal estrogen action in myometrium. Furthermore, estrogen appears a dominant regulator of both receptors in the myometrium.     

Regulation of progesterone receptor mRNA by oestradiol and antioestrogens in breast cancer cell lines

The induction of progesterone receptor mRNA by oestradiol and antioestrogens has been characterised in the MCF-7 breast cancer cell line. Progesterone receptor mRNA was induced more than 100-fold by oestradiol. The induction was half-maximal in the presence of 10(-10) M oestradiol and maximum levels were reached after 24 h treatment. Progesterone receptor mRNA was induced to 10% of the oestrogen-induced level by tamoxifen and its metabolite 4'-hydroxytamoxifen. The increase was half-maximal in the presence of 5 X 10(-8) M tamoxifen or 5 X 10(-10) M 4'-hydroxytamoxifen. In contrast, neither the benzothiophene antioestrogen LY117018 nor the 7 alpha-alkyl steroidal antioestrogen ICI 164,384 had any effect on progesterone receptor mRNA. The progesterone receptor mRNA was also induced by oestrogen in a T47D subline and in two other oestrogen-responsive breast cancer cell lines (ZR-75, EFM-19). Tamoxifen was a partial oestrogen for progesterone receptor mRNA induction in each of these cell lines. The large induction of the progesterone receptor mRNA by oestrogen in all 4 breast cancer cell lines supports the contention that the progesterone receptor may be a good predictive marker of hormonal response in human breast cancer.     

Molecular changes associated with the acquisition of oestrogen hypersensitivity in MCF-7 breast cancer cells on long-term oestrogen deprivation

The growth dependence of many breast cancers on oestrogen has been exploited therapeutically by oestrogen deprivation, but almost all patients eventually develop resistance largely by unknown mechanisms. Wild-type (WT) MCF-7 cells were cultured in oestrogen-deficient medium for 90 weeks in order to establish a long-term oestrogen-deprived MCF-7 (LTED) which eventually became independent of exogenous oestrogen for growth. After 15 weeks of quiescence (LTED-Q), basal growth rate increased in parallel with increasing oestrogen sensitivity. While 10⁻⁹ M oestradiol (E2) maximally stimulated WT growth, the hypersensitive LTED (LTED-H) were maximally growth stimulated by 10⁻¹³ M E2. By week 50, hypersensitivity was apparently lost and the cells became oestrogen independent (LTED-I), although the pure antioestrogen ICI182780 still inhibited cell growth and reversed the inhibitory effect of 10⁻⁹ M E2 at 10⁻¹² to 10⁻⁷ M. Tamoxifen (10⁻⁷ to 10⁻⁶ M) had a partial agonist effect on WT, but had no stimulatory effect on LTED. Whilst LTED cells have a low progesterone receptor (PgR) expression in all phases, oestrogen receptor (ER) a expression was, on average, elevated five- and seven-fold in LTED-H and LTED-I, respectively, and serine118 was phosphorylated. ERβ expression was up-regulated and the levels of insulin receptor substrate 1 (IRS-1) remained low throughout all phases. The levels of RIP140 mRNA appeared to decrease to approximately 50% of the WT message in LTED-Q and remained constant into the hypersensitive phase. No significant changes were observed in the expression of SUG-1, TIF-1 and SMRT in LTED. The overall changes in nuclear receptor interacting proteins do not appear to be involved in the hypersensitivity. Thus, the resistance of these human breast cancer cells to oestrogen-deprivation appears to be due to acquired hypersensitivity which may be explained in part by increased levels of and phosphorylated ER.     

Oestrogen receptor gene structure and function in breast cancer

The mechanisms underlying loss of oestrogen responsiveness in breast cancer are not well-defined. Potential mechanisms include loss of receptor expression, alterations in the oestrogen receptor (ER) gene producing proteins with abnormal function, or changes to receptor-dependent or -independent pathways controlling cell proliferation. Examination by Southern analysis of the ER gene in a series of ER-negative and -positive breast tumour biopsies failed to provide evidence of gross rearrangements and in only only one of thirty seven tumour DNA samples was significant gene amplification observed. No restriction fragment length polymorphisms were detected for the restriction enzymes EcoRI, Pst I or Hind III. Methylation of the ER gene as assessed by Hpa II and Msp I restriction enzyme digests varied between tumours but the degree of methylation was not correlated with levels of expression of the receptor protein. Similar findings applied in a series of ER-negative and -positive breast cancer cell lines and clonal lines of MCF-7 cells, which were developed as an in vitro model for the acquisition of oestrogen and antioestrogen resistance. In this model there was no evidence that changes to ER receptor function and/or structure at the level of the ER gene, mRNA, ligand binding, and ability to induce progesterone receptor might account for the development of hormone resistance. However, the ability of ER to interact with a DNA sequence containing the vitellogenin promoter oestrogen response element, as assessed by gel retardation assay, was impaired in the clone showing the greatest degree of oestrogen and antioestrogen resistance.     

ChIP‐Seq of ERα and RNA polymerase II defines genes differentially responding to ligands

We used ChIP‐Seq to map ERα‐binding sites and to profile changes in RNA polymerase II (RNAPII) occupancy in MCF‐7 cells in response to estradiol (E2), tamoxifen or fulvestrant. We identify 10 205 high confidence ERα‐binding sites in response to E2 of which 68% contain an estrogen response element (ERE) and only 7% contain a FOXA1 motif. Remarkably, 596 genes change significantly in RNAPII occupancy (59% up and 41% down) already after 1 h of E2 exposure. Although promoter proximal enrichment of RNAPII (PPEP) occurs frequently in MCF‐7 cells (17%), it is only observed on a minority of E2‐regulated genes (4%). Tamoxifen and fulvestrant partially reduce ERα DNA binding and prevent RNAPII loading on the promoter and coding body on E2‐upregulated genes. Both ligands act differently on E2‐downregulated genes: tamoxifen acts as an agonist thus downregulating these genes, whereas fulvestrant antagonizes E2‐induced repression and often increases RNAPII occupancy. Furthermore, our data identify genes preferentially regulated by tamoxifen but not by E2 or fulvestrant. Thus (partial) antagonist loaded ERα acts mechanistically different on E2‐activated and E2‐repressed genes.