Effects of Estrogen on Global Gene Expression:
Identification of Novel Targets of Estrogen Action1
1 Supported in part by: Susan G. Komen Breast Cancer Foundation, postdoctoral award to A.H.C. and NIEHS Center ES07784
Abstract
The important role played by the sex hormone estrogen (E2) in disease and physiological processes has been well documented. However, the mechanisms by which this hormone elicits many of its normal as well as pathological effects are unclear. In order to identify both known and unknown genes which are regulated by or associated with E2 action we performed serial analysis of gene expression (SAGE) on E2 responsive breast cancer cells after exposure to this hormone. We examined approximately 190,000 mRNA transcripts and monitored the expression behavior of 12,550 genes. Expression levels for the vast majority of those transcripts were observed to remain constant upon E2 treatment. Only approximately 0.4% of the genes showed an increase in expression of three fold or more by 3 h post E2 treatment.
We cloned five novel genes (E2IG1-5) which were observed up-regulated by the hormonal treatment. Of these the most highly induced transcript, E2IG1, appears to be a novel member of the family of small Heat shock proteins (HSPs).
The E2IG4 gene is a new member of the large family of leucine rich repeat-containing proteins. Based on architectural and domain homology this gene appears to be a good candidate for secretion in the extracellular environment and therefore may play a role in breast tissue remodeling and/or epithelium-stroma interactions.
Several interesting genes with a potential role in regulation of cell cycle progression were also identified to increase in expression including PES1 and Chaperonin CCT2.
Two putative paracrine/autocrine factors of potential importance in regulation of breast cancer cells growth were identified to be highly up-regulated by E2: Stanniocalcin 2 (STC2) a calcium/phosphate homeostatic hormone, and Inhibin Beta B (INHBB), a TGF beta like factor.
Interestingly, we also determined that, E2IG1 and STC2, were exclusively overexpressed in E2 receptor positive (ER+) breast cancer lines and thus they have potential to serve as breast cancer biomarkers.
This data provides a comprehensive view of estrogen's induced changes on the transcriptional program of human E2 responsive cells and it also identifies novel and previously unsuspected gene targets whose expression is affected by this hormone.
Introduction
The sex steroid hormone estrogen plays an essential role in the development of various tissues and in the maintenance of numerous physiological processes. However, it has also been well documented that estrogen plays a critical role in the etiology and progression of human breast and gynecological cancers (1).
It is known that the effect of estrogen is mediated through its ability to bind the ERs, alpha and beta, which are basically ligand activated transcription factors. Recently, numerous ER associated proteins, co-activators and repressors have been identified which are of importance in regulating the ER interaction with the basal transcription machinery (2). The mitogenic effects of estrogen, have been in large part attributed to its ability to increase the expression of key cell cycle regulatory genes (3). However, regulation of cell proliferation is just one aspect of interest in estrogen studies. Of much importance is the identification of "novel" downstream E2 effectors, regardless of their association with proliferation. Furthermore, the potential exists for such newly identified E2 targets to become biomarkers of relevance in the monitoring of estrogen-related disease conditions such as breast cancer and osteoporosis.
In this report we describe a comprehensive gene expression analysis of the effects of E2 using a classical E2 responsive human model. In order to perform the comparative gene expression profiling we used SAGE, a powerful global gene expression technique that allows for the quantitative evaluation of all cellular messenger RNA populations (i.e., the transcriptome) (4). As a result of this study various novel E2 targets, both known and previously unknown, were identified which will stimulate further studies into the mechanisms of E2 action.
Materials and Methods
Human Breast Cancer Cell Lines. The MCF-7 cell line batch used in these studies is directly derived from the original MCF7 cell line. The ability of E2 to induce cell proliferation was verified by Fluorescence Activated Cell Sorting (FACS) analysis and cell counts. MCF-7 cells were maintained in Iscove's minimal essential media (IMEM, Biofluids) without phenol red and supplemented with glutamine, 50µ/ml gentamycin and 10% fetal bovine serum (FBS, BioWhittaker). Other breast cancer cell lines used included; ZR-75-1, SKBR3, MDA-MB-157, MDA-MB-435, MDA-MB-453 and T47D grown in10%FBS/ DMEM and UACC-812 grown in 10%FBS/L-15. We also used normal human mammary epithelial cells (HME-87), normal mammary organoids (B43) and normal bulk breast tissues (HMG), as previously described (5).
SAGE. To obtain mRNA samples for SAGE, MCF-7 cells were seeded into 150mm plates (1.5 x 106 cells/plate) and allowed to reach a logarithmic growth phase in culture media supplemented with FBS. At 40% confluency cells were incubated for 48 h in culture media supplemented with 10% charcoal stripped serum (CSS, Hyclone). Cells were then treated with 10-8M 17ß-estradiol (SIGMA) or vehicle control (equivalent amounts of ETOH). Samples were collected at time 0 (i.e. untreated), 3 and 10 h after starting treatment. Total RNA was isolated using Qiagen total RNA Maxi kit (Qiagen) and mRNA was purified using Oligotex mRNA mini kit (Qiagen) following manufacturer's protocol. SAGE library generation and sequencing was performed as previously described by Velculescu et. al. (4). Statistical analysis of and comparison between different time points and controls were performed as described by Zhang et. al. (6) and using statistical functions available in the SAGE 3.0. software (kindly provided by Dr. K. Kinzler, Johns Hopkins University) for p chance calculations and Monte Carlo simulations.
Northern Blot Analysis. Samples for Northern Blot Analysis were obtained from MCF7 cells handled as described for SAGE and treated with 10-8M E2 for 0, 3, 6, 10, 15, 24 h and vehicle control for 24 h before harvesting. RNA isolation and Northern blotting was performed following standard procedures. Probes for hybridization were obtained by RT-PCR from breast cDNA libraries and their sequence was confirmed prior to use. Expression of ER alpha mRNA was confirmed using RT-PCR.
Cloning and Computer Analysis of Novel Transcripts. Novel transcripts were cloned from a Human Placenta cDNA library (Rapid Screen; Origene Technologies). To screen this library we used the same primers as for generating Northern probes. Each cDNA clone was sequenced in its entirety and the SAGE tag confirmed. The predicted amino acid sequence for each transcript was analyzed using BLASTP and PSIBLAST algorithms and the identification of protein family domains was determined using the Pfam domain models (PFAM). For protein cellular localization analysis we used the PSORT algorithm (7).
Results
Summary Of Global Gene Expression Findings. The SAGE method generates short sequences (i.e., transcript tags) specific to each expressed gene. The proportion of each tag in the overall tag population is representative of the proportion of each mRNA in the original mRNA population. Expression patterns are then deduced from the abundance of individual tags within each sample set. In the following study we generated a SAGE database of E2 responsive MCF-7 breast cancer cells treated with 10-8M 17ß-estradiol. The MCF-7 E2 dependent system was chosen because it is one of the best and most widely used models for the study of E2's effects on human cells (8).
MCF-7 cells were cultured in conditions, devoid of E2 for 48 h prior to the start of the experiments. Messenger RNA was then collected for SAGE at time points of 3 (E2 3h) and 10 (E2 10h) hours after E2 treatment. The sampled time-points allowed us to analyze changes occurring in the MCF7's transcriptome prior to entry into S phase, as determined by FACS (data not shown).
The SAGE profiles, for E2 treated cells (E2 3h and E2 10h), were compared with SAGE profiles from untreated MCF-7 cells (E2 0h) and a 3 h vehicle treated control. A total of 188,367 transcript tags (approximately 61,000 tags for each E2 0h, E2 3h and E2 10h) plus additional tags from vehicle control were sequenced and analyzed. These tags identified a total of 12,550 different transcripts. Of these, the vast majority (83%) showed matches in GenBank (GB) databases to either known genes or anonymous expressed sequence tags (ESTs). The remaining 17% of the tags (i.e. 2,100 transcripts) showed no database matches.
Comparison of the SAGE tag libraries from MCF7 treated and untreated cells demonstrated a remarkable similarity between expression profiles. Figure 2A illustrates scatter plots representing the relative expression of all transcripts analyzed, derived from pair-wise comparisons of the three SAGE libraries. The excellent correlation coefficients illustrates both, the reproducibility of SAGE using three different samples and RNA isolates, as well as the close similarity of the samples under analysis in this particular study. The vast majority of transcripts did not change in expression, 81.4% of them (approximately 10214 transcripts) exhibited less than a 2 fold difference in expression upon E2 treatment (Figure 2B).
Comparative statistical analyses of the tag libraries was performed to estimate the relative likelihood that a detected difference in expression would be seen by chance for each individual tag given the size of the SAGE libraries under study (6). Only 50 transcripts demonstrated an increase in expression at approximately 3 fold or greater at the p < 0.001 level of significance. Of these transcripts, 37 tags identified known genes, 8 corresponded to anonymous EST clusters, 2 match more than one sequence and 3 transcripts showed no reliable matches in the GB databases (Table 1). One transcript (arginino succinate synthase gene) was also observed to increase in the vehicle control treated cells.
The SAGE database containing the complete list of the 12,550 transcripts identified in the MCF7 cells, as well as their relative levels of expression under the various conditions of E2 treatment can be viewed at our website (http://sciencepark.mdanderson.org/ggeg). This MCF7 SAGE database displays, among other features, the p chance values for each individual comparison in relative transcript expression and active "tag links" connected to the recently described SAGEmap NCBI databases in which expression levels of each tag in various tissues and cell lines can be evaluated (9).
Cloning And Characterization Of Novel E2 Induced Genes. As indicated, several of the transcript tags shown in Table 1 identified anonymous EST clusters or had no matches in GB databases. On our follow-up studies to the SAGE analysis we first focused on those transcript tags which increased greater than 10 fold upon E2 treatment. As a result we isolated a series of five novel cDNAs from a human placenta cDNA library using transcript specific PCR primers. We named these novel genes E2IGs 1-5 for E2 Induced Genes. Full-length cDNA sequences for each of the novel transcripts have been reported to NCBI's GenBank databases under accession numbers AF191017, AF242180, AF191018, AF191019 and AF191020 for E2IG1, E2IG2, E2IG3, E2IG4 and E2IG5, respectively.
We observed that the SAGE tag CCTGGCCTAA increased in number from 0 to 18 tags by 3 h post E2 exposure in MCF7 cells. The gene which we cloned from this tag was named E2IG1. The corresponding cDNA is 2,007bp long containing a predicted open reading frame (ORF) of 588bp. The central portion of the 196AA protein encoded by the E2IG1 transcript is homologous to a highly conserved HSP alpha crystalline domain common to all HSP20 family members (10) (Figure 3A). Furthermore, E2IG1 shows a 54% homology to HSP27 suggesting that E2IG1 is a novel member of the small HSP family.
Based on a perfect match between the sequence of E2IG1 and the genomic sequence identified in GB as sts TIGR-A002J47, we can predict that this gene maps to chromosome 12 (D12S366 - D12S340 interval).
A second gene cloned, which is identified by SAGE tag GTCAGATGTC, was named E2IG2. This cDNA contains an ORF 291bp long encoding for a putative 97AA protein. Amino Acid Sequence Analysis identified some homology to two small proteins found in yeast (accession no. AAB67446 and CAA19105) and one small polypeptide found in Drosophila (accession no. AAF54887). We could not identify any known functional and/or structural domains within E2IG2's AA sequence. We can predict that E2IG2 maps to human chromosome 11 due to matching sequence information with sts T99047 (D11S916 - D11S911 interval).
The E2IG3 cDNA transcript corresponding to SAGE tag AGCTGTGTAA is 2,038bp long. This cDNA encodes for a 560AA polypeptide. Analysis of E2IG3's AA sequence indicates the existence of three overlapping putative nuclear localization signals at its N terminus and a strong GTP binding site towards its carboxyl end. This hypothetical GTP binding protein is predicted (83% probability) (7) to be located within the cell's nucleus. Interestingly, E2IG3 demonstrates 50% amino acid and structural homology to a nucleolar GTP binding protein, immunoreactive autoantigen Ngp-1(accession no. Q13823), found to be expressed in breast carcinomas . Based on analysis of on going human genome sequencing data we can also predict that E2IG3 maps to chromosome 3 since we found perfect matching to sequences reported in the GB entries AC016490 and AC012522.
The fourth gene cloned, E2IG4 corresponds to SAGE tag GGCATCAGGG which increased from 0 tags to 12 tags by 3 h post E2 exposure in MCF7 cells. This cDNA is 2,542bp long, showing a 1059bp ORF encoding for a 353 AA protein. This protein is characterized by the presence of a Leucine rich repeat in the N-terminal domain (LRRNT) and eight additional leucine rich repeats (Figure 3B). Interestingly, E2IG4 demonstrates domain and structural homology to extracellular matrix, leucine rich proteoglycans proteins such as Decorin (PGS2) and Biglycan (PGS1). The E2IG4 protein is likely to reside in the cell membrane or extracellular compartments (57% probability) (7). E2IG4 also contains a typical cleavable signal peptide at AA 1-16, which makes it a candidate protein for intracellular transport and extracellular secretion. In addition E2IG4 contains various potential phosphorylation sites throughout its amino acid sequence. Based on matching DNA sequence from sts-AA009735 to E2IG4, it is possible to predict the mapping of this gene also to human chromosome 11, (D11S911 Ð D11S4172 interval).
Finally E2IG5 whose identifying SAGE tag was found to increase mostly 10h post E2, consists of a 800bp long transcript. This cDNA demonstrates a 444bp ORF encoding for a 148 AA protein. Significant homology (81%) was found between E2IG5 and a growth and transformation dependent putative rat protein (pIL2 hypothetical rat protein, accession no. AAA42232). Interestingly and in possible agreement with our observations, the mRNA encoding for pIL2 was found to accumulate following growth-factor-induced transition of normal rat fibroblasts from a quiescent to a proliferative state (11). Analysis of the AA sequence of E2IG5 did not identify any conserved protein motif when comparing with existing protein databases. It was only possible to identify a putative transmembrame segment (AA102-122). Based on matching DNA sequence information between E2IG5 and GenBank Accession AC026269 we can predict that this gene also maps to human chromosome 11.
E2 Induced Expression Of Previously Known Genes
Chaperones. In addition to the novel genes described above we show in Table 1 all genes that were found to increase in expression at or above 3 fold with E2 treatment at the p < 0.001 level of significance. Among these transcripts we found abundant representation of protein members of a large class of molecular chaperones known as heat shock proteins (HSPs). In our studies, the most significant increases in gene expression were observed for HSP90 (both alpha and beta chains), HSP60 (Chaperonin), Chaperonin containing t-complex (CCT2), HSC71 (HSP70 family), FKBP4 (immunophilin/p59/HSP56) and the E2 induced gene described in this report, E2IG1 (HSP20 family) (Table 1).
Cell Cycle Progression Related Genes. SAGE tags identifying the Chaperonin containing t-complex gene (CCT2) were found to increase from 0 to 10 tags by 10 h post E2 treatment. This chaperone complex plays a fundamental role in cell cycle progression since it is in charge of the proper folding and maturation of Cyclin E molecules (12).
Among well known critical cell cycle targets of E2, the tags identifying Cyclin D1 were observed to increase from 32 to 113 SAGE tags (i.e. 3.5 fold) by 3 h of hormonal treatment.
The tag identifying the human homolog to the Zebrafish developmental gene Pescadillo (PES1) also demonstrated a marked increase in expression after E2 treatment (from 0 to 14 tags, Table 1). PES1 is predicted to encode a protein of 582 AA that is highly conserved from yeast to humans (13). Based on the presence of a BRCT (BRCA1 Carboxyl Terminus) domain in PES1, we can speculate that PES1 may be of relevance in cell cycle regulation (14).
Ran/TC4 another transcript up-regulated by the treatment, is a small GTP binding protein, member of the Ras superfamily, which is essential for the translocation of RNA and proteins through the nuclear pore complex (NPC). This GTPase may play a role in releasing steroid receptors from the NPC and in connecting DNA synthesis with the onset of mitosis (15, 16).
Tags identifying other relevant cell cycle progression modulators include those identifying Calmodulin subunits, CALM1 and CALM2. CALM1 expression increased from 30 to 50 tags (data not shown) and CALM2 increased from 12 to 40 tags (p < 0.001) upon E2 treatment (Table 1). It is known that this important calcium binding protein plays an essential role for quiescent cells to enter the cell cycle (17). Calmodulin also regulates phosphorylation and induces conformational changes of the ER (18, 19).
We also observed increase in tag numbers identifying subunits of the Proteasome complex, a multicatalytic intracellular protease system which targets key proteins for degradation (20). The ubiquitin-proteasome pathway also appears to be a major mechanism implicated in the turn-over of the ER in an E2 - dependent manner which would agree with our findings (21).
Paracrine-Autocrine Factors. Among interesting gene targets not previously associated with E2 effects, we observed a ten fold increase in expression for stanniocalcin2 (STC2) (Table 1). STC2 (also known as Stanniocalcin related peptide STCrP) has amino acid sequence homology to the previously identified STC1 (22, 23). However, the SAGE tag matching STC1 did not increase in numbers nor did we observed upregulation by Northern analysis (data not shown).
An additional autocrine/paracrine factor, Inhibin beta-B subunit (Activin beta-b subunit) (INHBB) was identified to increase above 15 fold upon E2 treatment (Table 1). Originally Inhibin was identified as a gonadal hormone that inhibits the secretion of FSH (whereas Activin stimulates the secretion of FSH) by the pituitary gland (24). Activins /Inhibins belong to the TGF - beta superfamily (25). The only Inhibin related transcript tag found upregulated by E2 treatment was that of INHBB subunit.
Tumor Associated Genes. SAGE identified an increase in expression for various tumor associated proteins, RFP (ret finger protein), D52L1 (D53 tumor protein), Trefoil factor (TFF1 or PS2), Caveolin 1 (CAV1) and nm23H1 (NDKA) among others (Table 1).
RFP was first identified for its elevated expression in a variety of rodent tumor cell lines (26). This protein is located in the nucleus and because of its zinc finger domain is believed to bind DNA. However the function of this protein is, at present, unknown.
Tumor protein, D52L1 also known as D53, increased from 7 tags in untreated cells to 27 tags in the 3 h E2 treatment profile. D53 was originally cloned from a breast carcinoma cDNA library and has a 52% homology to D52 (27). Interestingly and in agreement with our SAGE findings, previous studies have shown that both, D52 and D53 transcription in breast carcinoma cells, are dependent upon E2 presence in MCF7 cells (27).
The well known E2 regulated gene PS2 (Trefoil factor) (28) was also detected to increase 14 fold by SAGE from 13 tags at 0h to 186 tags by 10 h post E2 treatment.
Interestingly, we observed a significant increase in tags identifying the putative metastasis suppressor gene nm23H1 (2.5. fold increase, p value = 0.000003), also known as NDKA (Nucleoside Diphosphate Kinase A). It is likely that such increase in expression is related to its fundamental housekeeping role in maintaining the NDP/NTP cellular balance (29).
The tags identifying Caveolin 1 (CAV1) were also shown to increase about 4 fold (Table 1). CAV1 is an integral membrane protein and the main component of caveolae membranes (a plasma membrane specialization). It was recently shown that CAV1 functions as a membrane adaptor to link integrin subunits to the tyrosine kinase FYN (30).
Validation Of SAGE Findings. The effects of E2 treatment on the expression of specific genes were confirmed by means of Northern analysis. To this end MCF-7 cells were grown in conditions devoid of E2 for 48 h and then exposed to E2 treatment various lengths of time as shown in Figure 5. These representative transcripts demonstrated different patterns of expression. Some transcripts increased and remained high for the entire course of treatment whereas others increased rapidly in early time points and then tapered off in later time points (Figure 5). As can be observed, E2IG1 and E2IG4, increased considerably already by 3 h of E2 treatment. E2IG5 showed only a modest increase in expression levels as detected by total RNA Northern blot analysis (data not shown). The reason for the apparent discrepancy between the fold values of increase expression detected by SAGE versus those detected by Northern analysis in this last case are unclear.
It is worth mentioning that Northern blot analysis also confirmed SAGE observations in transcripts which demonstrated a less than 3-fold difference. For example for the Nm23H1 transcript SAGE detected 27 tags in control compared to 67 tags in the E2 treated (Table 1) and Northern blot analysis confirmed this fold difference with remarkable accuracy to a 2.5 fold change. In addition SAGE also identified a 3.5 fold increase in Cyclin D upon E2 treatment, in close agreement with previously reported Northern and Western data (3).
Transcript Expression In ER+ And ER- Breast Cancer Cell Lines. Northern blot analysis was also used to assess the level of expression for E2IG1, E2IG2, E2IG3, E2IG4 and STC2 in a small panel of estrogen receptor positive (ER+) and estrogen receptor negative (ER-) cell lines. The expression of estrogen receptor alpha for each of the cell lines was confirmed by RT-PCR (Figure 6). Both E2IG1 and STC2 demonstrated selective expression in ER+ cell lines (Figure 6) whereas E2IG2, E2IG3 and E2IG4 demonstrated varied expression in the different cell lines (data not shown). The selective expression in ER+ cell lines for STC2 and E2IG1 suggests their expression is dependent on the presence of a functional estrogen receptor.
Discussion
In this study we have used a powerful global gene expression methodology in order to identify novel direct or associated targets of estrogen action. Our studies led us to identify and clone five novel genes shown to increase upon E2 treatment. We also identified a series of previously unsuspected targets of E2 effects.
Our cloning effort was focused on those SAGE tags identifying anonymous ESTs which increased above 10 fold upon E2 treatment. Among these, E2IG1 was identified as a putative small HSP and which bears 54% homology to HSP27. Interestingly, the HSP27 expression has been associated with the presence of ER in breast and endometrial carcinomas (31). It was remarkable in our study, that there is selective over-expression of E2IG1 in ER+ breast cancer cell lines (Figure 6). These finding suggests that E2IG1 expression is dependent on the presence of a functional ER. Based on these observations and on the rapid and dramatic up-regulation of E2IG1 after E2 treatment, we can speculate that this gene is a prime candidate to be a direct effector of estrogen. Since E2IG1 is a putative new member of the small HSP family, it may play a specific chaperonic role related to either the ER itself or some point downstream of the E2 - induced signaling cascade. In addition, the described selective over-expression of E2IG1 in ER+ breast tumor cells make it a good potential marker for E2 dependent breast carcinomas.
A second very intriguing novel target of E2 treatment is the E2IG4 protein. This putative protein is a new member of the large family of leucine rich repeat- containing proteins. Based on sequence homology and motif distribution analysis is possible to speculate that E2IG4 may constitute a novel extracellular matrix component. Analysis of E2IG4 amino acid sequence showed some moderate homology to GAC1, a leucine rich protein amplified in gliomas (32). E2IG4 also showed domain and leucine repeat distribution homology, as well as general features similarity, to several cleaved extracellular proteins such as GPV (Platelet glycoprotein V precursor), to PGS2 (Bone proteoglycan II precusor) also known as Decorin and to PGS1 (Biglycan). Both Decorin and Biglycan belong to the small interstitial proteoglycans family and are found in the extracellular matrix. Interestingly, in rat, Decorin has been isolated from cervix uteri and it appears to be hormonally regulated (33).
The presence of a typical signal peptide cleavage site in E2IG4's AA sequence also makes this protein a good candidate for transport to the cell membrane and secretion to the extracellular environment. The combination of the described E2IG4 structural features and homologies plus the observed up-regulation induced by estrogen suggests that this protein could play a role in hormonally regulated, extracellular matrix remodeling and/or epithelium-stromal interactions in breast tissue.
Among the SAGE tags increasing in abundance upon E2 treatment, several identified various members of large families of proteins with chaperonic function. Although the induced expression of HSPs is not unique to E2 and, there is evidence to suggest that the expression of several HSPs may play a role in breast cancer and E2 regulation (34-36). HSP90, also functions as a stabilizer of critical signal transducers including cell cycle and developmental regulators. These include Src-family-kinases, raf serine/threonine kinases, calmodulin, dioxin-receptor, cyclin- dependent kinases and steroid-hormone receptors (37). HSP90 together with HSP70 play important roles in maturation of the steroid receptor to achieve a hormone-binding competent state as well as regulating the receptor cytoplasmic-nuclear trafficking (15). The HSP90 binding protein, FKBP4, was also observed increasing in expression upon E2 treatment. Both HSP90 and FKBP4 are known to bind unliganded, steroid receptor complexes (38).
Interestingly SAGE also identified up-regulation of CCT2, a chaperone protein which has recently been shown to bind newly synthesized Cyclin E, mediating its maturation (folding) into a form that can associate with Cdk2 (12). The important function of Cyclin E as a key player promoting the G1 to S phase transition in breast cancer cells has been shown (39). The demonstrated ability of CCT2 to in turn control the maturation of Cyclin E and hence its critical function in the cell cycle appears to provide an additional layer of influence in cell cycle progression by E2.
PES1 was another gene observed to be up-regulated upon E2 treatment which also may play a role in cell cycle regulation. The highly conserved PES1 displays a region of approximately 100 amino acids (322-415) with homology to the BRCT domain superfamily. BRCT domains are usually found in proteins that play critical roles in cell cycle control and DNA repair (14). The possibility that PES1 is playing a role in either of these processes is of interest and therefore deserves further investigation.
Among the identification of novel targets of estrogen action it is important to stress the dramatic increase in expression observed for STC2. Although stanniocalcin (STC) was initially thought to be unique to fish, the two mentioned homologues of STC have now been identified in mammalians (i.e., STC1 61% homology and STC2 30-38% homology). In humans, the function of these hormones is unknown. However in fish it was observed that STC functions as a potent regulator of calcium and phosphate homeostasis preventing hypercalcemia in a similar fashion to Calcitonin in mammals (40). It has been proposed that mammalian stanniocalcins act as regulators of calcium/phosphate homeostasis in a paracrine and/or autocrine rather than endocrine fashion, as suggested by their widespread expression in tissues (41-43). Some evidence suggests that STC2 acts in a manner opposite to STC1 on regulating calcium/phosphate concentrations (23). The fact that STC2 appears to be secreted protein and is selectively over-expressed in ER+ cell lines suggest that it may have potential use as a serum detectable prognostic marker for breast cancer. In addition, due to its potential role as a calcium/phosphate regulator and its wide expression including in bone, STC2 becomes a prime target for further study since it may provide a novel link between the effects of estrogen and bone remodeling.
SAGE also identified another interesting E2 regulated, autocrine/paracrine factor INHBB. This protein subunit functions either as a homodimer (Activin B) or a heterodimer with beta-A or alpha subunits (forming Activin AB or Inhibin B respectively). The elevation of Inhibin subunits, alpha and beta B, after E2 treatment has been observed in granulosa cells (44). It has also been shown that ER+ breast cancer lines are growth inhibited by Activin B (45). Furthermore, the expression of Inhibin/Activin subunits and corresponding receptors has been detected in MCF7 cells (46). Theoretically the increased expression of INHBB would tilt the homo/heterodimer balance toward generation of Activin B, since INHBB was the only inhibin subunit found to increase upon E2 treatment. This appears to point to a paradoxical effect of E2 since Activins along with the rest of the TGF-beta family are thought to contribute to maintaining a negative growth regulatory function in ER+ breast cancer cells which would be in opposition to estrogen's ability to stimulate cell growth (45). Further study is therefore required in order to better understand the role of modifications in the Activin/Inhibin homeostasis in E2 responsive breast cancer cells and its putative effect in regulating cell growth.
In this report we have summarized some of the most prominent effects induced by E2 affecting the transcriptional program of estrogen responsive cells at a global level. As mentioned, several of the observed changes in gene expression will be common to those induced by other mitogens. On the other hand numerous changes observed are likely to be directly induced by this important female steroid hormone. Follow up studies on the mechanisms of expression regulation for the novel targets described as well as for previously unsuspected effectors of estrogen are now in order.
It is also important to stress that several of these genes have a very good potential to serve as diagnostic/prognostic tools for the monitoring of estrogen related disease conditions such as breast cancer.