Abstract
The important role played by the sex hormone estrogen 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. To identify both known and unknown genes that are regulated by or associated with estrogen action, we performed serial analysis of gene expression on estrogen-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 17β estradiol (E2) treatment. Only approximately 0.4% of the genes showed an increase in expression of ≥3-fold 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. The E2IG4 gene is a new member of the large family of leucine-rich repeat-containing proteins. On the basis of 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 the regulation of cell cycle progression were also identified to increase in expression, including Pescadillo and chaperonin CCT2. Two putative paracrine/autocrine factors of potential importance in the regulation of the growth of breast cancer cells were identified to be highly up-regulated by E2: stanniocalcin 2, a calcium/phosphate homeostatic hormone; and inhibin-β B, a TGF-β-like factor. Interestingly, we also determined that E2IG1 and stanniocalcin 2 were exclusively overexpressed in estrogen-receptor-positive breast cancer lines, and thus they have the potential to serve as breast cancer biomarkers. This data provides a comprehensive view of the changes induced by E2 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 E2 is mediated through its ability to bind the ERs, α and β, which are basically ligand-activated transcription factors. Recently, numerous ER-associated proteins, coactivators, and repressors have been identified that are of importance in regulating the ER interaction with the basal transcription machinery (2) . The mitogenic effects of E2 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 E2 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. 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 mRNA populations (i.e., the transcriptome; Ref. 4 ). As a result of this study, various novel E2 targets, both known and previously unknown, were identified, which will stimulate additional 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 derived directly from the original MCF-7 cell line. The ability of E2 to induce cell proliferation was verified by fluorescence-activated cell sorting analysis and cell counts. MCF-7 cells were maintained in Iscove’s MEM (Biofluids) without phenol red and supplemented with glutamine, 50μ g/ml gentamicin, and 10% 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 described previously (5) .
SAGE.
To obtain mRNA samples for SAGE, MCF-7 cells were seeded into 150-mm plates (1.5 × 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 (Hyclone). Cells were then treated with 10−8 m 17β-estradiol (Sigma) or vehicle control (equivalent amounts of ethanol). Samples were collected at time 0 (i.e., untreated), 3, and 10 h after starting treatment. Total RNA was isolated using the Qiagen total RNA Maxi kit (Qiagen) and mRNA was purified using the Oligotex mRNA mini kit (Qiagen) following the manufacturer’s protocol. SAGE library generation and sequencing was performed as described previously 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 by using statistical functions available in the SAGE 3.0 software (kindly provided by Dr. K. Kinzler, The John Hopkins University) for P calculations and Monte Carlo simulations.
Northern Blot Analysis.
Samples for Northern blot analysis were obtained from MCF-7 cells handled as described for SAGE and treated with 10−8 m E2 for 0, 3, 6, 10, 15, and 24 h and with 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 before use. Expression of ER-α 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 was 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. 4 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 10−8 m E2. The MCF-7 E2-dependent system was chosen because it is one of the best and most widely used models for the study of the effects of E2 on human cells (8) .
MCF-7 cells were cultured in conditions devoid of E2 for 48 h before the start of the experiments. mRNA was then collected for SAGE at time points of 3 (E2 3 h) and 10 (E2 10 h) h after E2 treatment. The sampled time points allowed us to analyze changes occurring in the transcriptome of the MCF-7 cells prior to entry into S phase, as determined by fluorescence-activated cell sorting (data not shown).
The SAGE profiles for E2-treated cells (E2 3 h and E2 10 h), were compared with SAGE profiles from untreated MCF-7 cells (E2 0 h) and a 3-h, vehicle-treated control. A total of 188,367 transcript tags (approximately 61,000 tags for each E2 0 h, E2 3 h, and E2 10 h) 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 GB databases to either known genes or anonymous ESTs. The remaining 17% of the tags (i.e., 2,100 transcripts) showed no database matches.
Comparison of the SAGE tag libraries from MCF-7 treated and untreated cells demonstrated a remarkable similarity between expression profiles. Fig. 1A ⇓ illustrates scatter plots representing the relative expression of all transcripts analyzed, derived from pairwise 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 (≈10,214 transcripts) exhibited a <2-fold difference in expression upon E2 treatment (Fig. 1B) ⇓ .
A, scattergrams comparing the levels of expression of the 12,550 transcripts detected in MCF-7 at time points 0, 3, and 10 h after treatment with 10−8 m E2. The Y and X axes indicate the total number of tags detected for each of the indicated conditions. Each white dot represents a transcript, and its location in the plot will depend on the relative expression in the conditions compared. As can be observed by the excellent r squares obtained, the three samples are remarkably similar, indicating that relatively few genes are found to be differentially expressed in a significant way. B, summary of the ratios of abundance of SAGE transcript tags expressed in MCF-7 cells in the presence (10-h time point) versus the absence of E2. To avoid division by zero, we used a tag value of 1 for any tag that was not detectable in one of the samples. Ratios were rounded to the nearest integer. Negative integers represent a decrease in tag numbers upon E2 treatment, and positive integers represent an increase in tag numbers upon treatment. The number of tags displaying each ratio is plotted on the Y axis.
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 at the P <0.001 level of significance. Of these transcripts, 37 tags identified known genes, 8 corresponded to anonymous EST clusters, 2 matched more than one sequence, and 3 transcripts showed no reliable matches in the GB databases (Table 1) ⇓ . One transcript (the arginino succinate synthase gene) was also observed to increase in the vehicle control-treated cells.
Transcripts induced by E2 treatment in MCF-7 cells
The SAGE database containing the complete list of the 12,550 transcripts identified in the MCF-7 cells, as well as their relative levels of expression under the various conditions of E2 treatment, can be viewed at our website. 5 This MCF-7 SAGE database displays, among other features, the Ps for each individual comparison in relative transcript expression and active “tag links” connected to the recently described SAGEmap National Center for Biotechnology Information 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 that increased >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 the GB databases of the National Center for Biotechnology Information under accession nos. AF191017, AF242180, AF191018, AF191019, and AF191020 for E2IG1, E2IG2, E2IG3, E2IG4, and E2IG5, respectively.
The gene cloned from tag CCTGGCCTAA, which increased in number from 0 to 18 tags, was named E2IG1. The corresponding cDNA is 2007 bp long containing a predicted open reading frame of 588 bp. The central portion of this 196AA protein is homologous to a highly conserved HSP-α crystalline domain common to all HSP20 family members (10) . Furthermore, E2IG1 shows a 54% homology to HSP27, suggesting that E2IG1 is a novel member of the small HSP family. On the basis of matching sequence with sts TIGR–A002J47, we can predict that this gene maps to chromosome 12 (D12S366–D12S340 interval).
E2IG4 corresponds to the SAGE tag GGCATCAGGG, which increased from 0 tags to 12 tags by 3 h post-E2 exposure in MCF7 cells. This cDNA is 2542 bp long, showing a 1059-bp open reading frame encoding for a 353AA protein. This protein is characterized by the presence of a leucine-rich repeat in the NH2-terminal domain and eight additional leucine-rich repeats (Fig. 2) ⇓ . Interestingly, E2IG4 demonstrates domain and structural homology to extracellular matrix leucine-rich proteoglycans such as decorin (PGS2) and biglycan (PGS1). The E2IG4 protein is likely to reside in the cell membrane or extracellular compartments (57% probability; Ref. 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. On the basis of matching DNA sequence from sts AA009735 to E2IG4, it is possible to predict the mapping of this gene to human chromosome 11, (D11S911–D11S4172 interval).
The E2IG4 transcript. The predicted amino acid sequence of the E2IG4 transcript is depicted, including leucine repeats (dark box), leucine-rich repeat in the NH2-terminal (light box), signal peptide (open box), and conserved amino acid sequences (bold). The last CATG and tag sequence are also depicted in bold.
The E2IG2 cDNA, encodes for a 97AA protein with some homology to two yeast proteins. The E2IG3 gene appears to be a nuclear 560AA GTP-binding protein and the E2IG5 cDNA encodes a 148AA protein with significant homology to a transformation-dependent rat protein (pIL2; Ref. 11 ). More specific information on these novel transcripts is available on our Internet site. 5
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 ≥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 HSPs. In our studies, the most significant increases in gene expression were observed for HSP90 (bothα and β chains), HSP60 (chaperonin), 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 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 because 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 homologue to the zebrafish developmental gene 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 582AA that is highly conserved from yeast to humans (13) . On the basis of the presence of a BRCT (BRCA1 COOH 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. This GTPase may play a role in releasing steroid receptors from the nuclear pore complex 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 an increase in tag numbers identifying subunits of the proteasome complex, a multicatalytic intracellular protease system that targets key proteins for degradation (20) . The ubiquitin-proteasome pathway also appears to be a major mechanism implicated in the turnover of the ER in an E2-dependent manner, which would agree with our findings (21) .
Paracrine-Autocrine Factors.
Among interesting gene targets not associated previously with E2 effects, we observed a 10-fold increase in expression for 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 observe up-regulation by Northern analysis (data not shown).
An additional autocrine/paracrine factor, INHBB (activin-β-b subunit) was identified to increase >15-fold upon E2 treatment (Table 1) ⇓ . Originally inhibin was identified as a gonadal hormone that inhibits the secretion of follicle-stimulating hormone (whereas activin stimulates the secretion of follicle-stimulating hormone) by the pituitary gland (24) . Activins/inhibins belong to the TGF-β superfamily (25) . The only inhibin-related transcript tag found up-regulated by E2 treatment was that of the 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), CAV1, and 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 MCF-7 cells (27) .
The well-known E2 regulated gene PS2 (Trefoil factor; Ref. 28 ) was also detected to increase 14-fold by SAGE from 13 tags at 0 h to 186 tags by 10-h-post-E2 treatment.
Interestingly, we observed a significant increase in tags identifying the putative metastasis suppressor gene NDKA/Nm23H1 (2.5-fold increase; P = 0.000003). It is likely that such an increase in expression is related to its fundamental housekeeping role in maintaining the nucleoside diphosphate/nucleotide triphosphate cellular balance (29) .
The tags identifying CAV1 were also shown to increase∼ 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 for various lengths of time as shown in Fig. 3 ⇓ . 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 (Fig. 3) ⇓ . As can be observed, E2IG1 and E2IG4 already increased considerably 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 increased expression detected by SAGE versus those detected by Northern analysis in this last case is unclear.
Validation of E2-induced expression of specific transcripts in MCF-7 cells. Representative Northern blots are shown for several transcripts identified by SAGE to be expressed at higher levels in E2-treated samples as compared with untreated (0 h) samples. MCF-7 cells were grown in conditions devoid of E2 for 48 h, and at time 0 cells were treated with 10−8 m E2 over a 24-h time course. Controls included MCF-7 cells maintained in the presence of FBS (first lane) and MCF-7 cells treated with equivalent amounts of vehicle (ethanol) for 24 h (24 control). All Northern analyses were performed on total RNA with the exception of INHBB, in which mRNA was used (bottom two rows). A probe for glyceraldehyde-3-phosphate dehydrogenase was used as a control for RNA loading.
It is worth mentioning that Northern blot analysis also confirmed SAGE observations in transcripts that demonstrated a <3-fold difference; e.g., for the NDKA transcript, SAGE detected 27 tags in control compared with 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 Northern and Western data reported previously (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 ER+ and ER- cell lines. The expression of ER-α for each of the cell lines was confirmed by RT-PCR (Fig. 4) ⇓ . Both E2IG1 and STC2 demonstrated selective expression in ER+ cell lines (Fig. 4) ⇓ , 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 ER.
Northern blot analysis of various breast cancer cell lines. ER+ cell lines including MCF-7, T47D, HME-87, UACC812, and ZR751 and estrogen ER- cell lines including MDA MB157, MDA MB435, MDA MB453, and SKBR3 were used for Northern blot analysis on the transcript E2IG1 and STC2. RT-PCR was used to confirm the expression of ERα, bottom panel.
Discussion
In this study we have used a powerful global gene expression methodology to identify novel direct or associated targets of E2 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 was selective overexpression of E2IG1 in ER+ breast cancer cell lines (Fig. 4) ⇓ . These findings suggests that E2IG1 expression is dependent on the presence of a functional ER. On the basis of 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 E2. Because 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 to some point downstream of the E2-induced signaling cascade. In addition, the described selective overexpression 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. On the basis of sequence homology and motif distribution analysis, it is possible to speculate that E2IG4 may constitute a novel extracellular matrix component. Analysis of the 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 of similarity, to several cleaved extracellular proteins such as: platelet glycoprotein V precursor; PGS2 (bone proteoglycan II precusor), also known as decorin; and 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 the AA sequence of E2IG4 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 E2 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. There is evidence to suggest that the expression of HSPs may play a role in breast cancer and in E2 regulation, however the induced expression of HSPs is not unique to E2 (34, 35, 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 the maturation of the steroid receptor in achieving a hormone-binding competent state as well as in 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 cyclin-dependent kinase 2 (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 control in turn 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 that 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 additional investigation.
Among the identification of novel targets of E2 action, it is important to stress the dramatic increase in expression observed for STC2. Although 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 STCs 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, 42, 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 a secreted protein and is selectively overexpressed in ER+ cell lines suggests that it may have potential use as a serum-detectable prognostic marker for breast cancer. In addition, because of its potential role as a calcium/phosphate regulator and its wide expression, including in bone, STC2 becomes a prime target for additional study because it may provide a novel link between the effects of E2 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 β-A or -α subunits (forming activin AB or inhibin B, respectively). The elevation of inhibin subunits α and β-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 MCF-7 cells (46) . Theoretically, the increased expression of INHBB would tilt the homo/heterodimer balance toward generation of activin B, inasmuch as INHBB was the only inhibin subunit found to increase upon E2 treatment. This appears to point to a paradoxical effect of E2 because activins, along with the rest of the TGF-β family, are thought to contribute to maintaining a negative-growth regulatory function in ER+ breast cancer cells, which would be in opposition to the ability of E2 to stimulate cell growth (45) . Additional study is therefore required 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 E2-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 E2, 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.
Footnotes
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵1 Supported in part by the Susan G. Komen Breast Cancer Foundation, postdoctoral award to A. H. C., and National Institute of Environmental Health Sciences Center Grant ES07784.
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↵2 To whom requests for reprints should be addressed, at University of Texas, M. D. Anderson Cancer Center, Science Park Research Division, P. O. Box 389, Smithville, TX 78957. Phone: (512) 237-9530; Fax: (512) 237-2475; E-mail: maldaz{at}odin.mdacc.tmc.edu
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3 The abbreviations used are: E2, 17β estradiol; FBS, fetal bovine serum; SAGE, serial analysis of gene expression; RT-PCR, reverse transcription-PCR; HSP, heat shock protein; CCT2, chaperonin-containing t-complex; STC, stanniocalcin; INHBB, inhibin-β B; ER, estrogen receptors; GB, GenBank; EST, expressed sequence tag; CAV1, caveolin 1; NDKA, nucleoside diphosphate kinase A, or Nm23H1; PES1, Pescadillo.
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↵4 Internet address: http://pfam.wustl.edu.
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↵5 Internet address: http://sciencepark.mdanderson.org/ggeg.
- Received June 16, 2000.
- Accepted September 15, 2000.
- ©2000 American Association for Cancer Research.
References
Volume 60, Issue 21
