The activity of many transcription factors is regulated by post-translational modifications such as phosphorylation, proteasome-mediated degradation and cross-talk with other signal transduction pathways. We will focus upon these three aspects in our discussion of ER function.
Stimulation of a number of growth factor receptors and/or protein kinases leads to the phosphorylation of ER resulting in ligand-independent and/or a synergistic increase in transcriptional activation in response to hormone (Kato et al, 2000).
The AF-1 region of ERa contains phosphorylation sites for a number of kinases including MAPK, cyclin A/cdk2 and PI3/AKT. In particular, much work has been focused on phosphorylation of serine residue 118 within the A/B domain of ERa. Phosphorylation of this particular residue by growth factor activated MAPK leads to the enhancement of the N-terminal AF-1 function. MAPK is activated by tyrosine kinase cell membrane receptors, that in turn are stimulated by growth factors such as insulin, IGF-1, EGF and TNF-a. Furthermore, phosphorylation at ER residue 118 also enhances the interaction of this receptor with the p68 RNA helicase, resulting in increased AF-1 but not AF-2 activity.
The phosphorylation of ER/3 by MAPK in cells treated with exogenous EGF or via overexpression of activated Ras has been shown to enhance binding between SRC-1 and the ER/3 AF-1 domain. This suggests that ligand-independent activation of ER/3 also depends on phos-phorylation of the N-terminal region, and that this event may be important for the recruitment of coactivators such as SRC-1.
Much less is known about the phosphorylation by the cyclin A/cdk2 or the PI3/Akt kinases and their effects on specific ER function. Specifically, phosphorylation of two serine residues at amino acid 104 and 106 of the ERa by the cyclin A/cdk2 enhances the ERa AF-1 function both in the absence of oestrogen or in the presence of tamoxifen. Furthermore, it was recently shown that PI3 kinase is able to increase both AF-1 and AF-2 activity, whereas Akt, a kinase downstream of PI3 kinase, increases only AF-1 activity. Phosphorylation of the ERa serine residue 167 by Akt also results in protection of breast cancer cells from tamoxifen-induced apoptosis, revealing an important potential mechanism for the onset of resistance to tamox-ifen in breast cancer.
Another major ligand-independent phosphorylation site within ERa is the conserved tyrosine 537 residue and the homologous tyrosine 443 residue in ER/3, but the exact consequence of phosphorylation at this site is still controversial and remains under intense investigation. Replacement of this tyrosine residue with other amino acids suggests that phosphorylation at this site is important for hormone binding and transcriptional activation. Specifically, the substitution of tyrosine 537 with alanine, as-paragine, or serine results in a mutant ERa that is constitutively active and binds to SRC-1 even in the absence of oestrogen. Another recent study also indicated that phosphorylation of the tyrosine 537 is critically involved in ligand-induced conformational changes of the ERa.
Recently, ubiquitin-dependent, proteasomal degradation of ligand-bound ERa was discovered as an additional mechanism involved in the regulation of hormone receptor-mediated gene transcription. The SRC coactivator family is also a target for degradation via the 26S protea-some. It is well known that the ubiquitin pathway is involved in the degradation of many short-lived proteins (Hershko and Ciechanover, 1998). Through a series of enzymatic reactions, ubiquitin is covalently linked to proteins targeted for degradation, marking them for recognition by the 26S proteasome, a large multisubunit protease. Abnormalities in ubiquitin-mediated degradation have been shown to cause several pathological conditions, including malignant transformation. In particular, it has recently been shown that ERa is ubiquitinated preferentially in the presence of hormone. It is thought that ERa protein degradation, which occurs through the 26S proteasome complex, is required for continued transcrip-tional activation by this receptor. ERa degradation could well be an important requisite to dissociate the preinitia-tion complex resulting in the release of the components necessary for another round of transcription. On the other hand, hormone-induced degradation may also serve as a negative feedback to down-regulate the transcription of oestrogen-responsive genes.
THE IMPORTANCE OF OESTROGEN RECEPTOR EXPRESSION IN CANCER INITIATION AND PROGRESSION
Oestrogen receptor gene expression in breast epithelium is an intricately regulated event, and is thought to play a central role in normal breast development, and also breast cancer evolution. ERa expression is significantly increased in both premalignant and malignant breast lesions, and many of these ERa-positive cells proliferate as compared with normal breast. Furthermore, normal breast epithelium, in addition to breast cancer tissue, contains alternatively spliced ERa and ERj3 mRNA variants, but it is still unclear whether changes in the levels of these variants impact upon tumour development or the progression to hormone-independent tumour growth. Single amino acid mutations within the ERa are relatively rare, but may contribute to the progression of breast cancer or metastatic disease. We will next describe the potential role of ERa expression in premalignant disease, as well as the role of specific ER variants and mutations in breast cancer development and progression.
Oestrogen Receptor Expression in Normal Breast and Breast Cancer
In normal nonpregnant, premenopausal human breast, only about 5-10% of the total luminal epithelial cell population expresses ERa, and this expression tends to be highest in the follicular phase of the menstrual cycle. The highest percentage of ERa-expressing cells are found in undifferentiated lobules type 1 (Lob1), with a progressive reduction in the more differentiated Lob2 and Lob3 types. The highest level of cell proliferation is also observed in Lob1, but expression of ERa occurs in cells other than these proliferating cells, indicating that they represent at least two separate cell populations. These data also suggest that oestrogen might stimulate ERa-positive normal cells to produce a growth factor that in turn stimulates neighbouring ERa-negative normal cells to proliferate. In pre-malignant and malignant breast lesions, however, ERa expression is significantly increased in the proliferating cell compartment, suggesting that ERa may be involved in the earliest changes to malignancy. Additionally, approximately two-thirds of breast tumours, at least initially, express abundant levels of ERa, and this expression is associated with lower risk of relapse and prolonged overall survival. Unfortunately, we still understand very little about the precise role of ERa expression in tumour progression.
Owing to its recent discovery, only limited data are available on the expression and function of ERft in normal breast and its potential role in breast carcinogenesis. Studies of ERft knockout mice suggest that ERft plays a limited role in normal breast development and function. However, ERft expression appears to be important for the growth control of urogenital tract epithelium, and may even afford a protective role against hyperproliferation and carcino-genesis in this particular tissue. This interesting hypothesis might also apply to the mammary gland, and is supported by a recent study reporting that ERft expression in breast tumours is positively associated with ERa and progesterone receptor expression, as well as negative axillary nodes, DNA diploidy and low S phase fraction, all of which imply that ERft-positive tumours may have a more favourable prognosis. On the other hand, two studies examining a relatively small number of tumours using RT-PCR determined that coexpression of ERft and ERa is frequently associated with poor prognostic biomarkers, such as positive axillary nodes and higher tumour grade, and also that ER is significantly elevated in tumours resistant to tamoxifen treatment.
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