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Molecular Oncology, Markers, Clinical Correlates |
Detection in Human Breast Tumors in Vivo
1 Manitoba Institute of Cell Biology, Departments of Biochemistry and Medical Genetics and 2 Pathology, University of Manitoba, Winnipeg, Manitoba, Canada
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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specifically phosphorylated at Ser118 is detectable in multiple human breast cancer biopsy samples. To gain insight into possible roles for P-Ser118-ER in human breast cancer in vivo.
Experimental Design: A specific antibody for P-Ser118-ER was validated for immunohistochemistry (IHC), and Western blot analysis confirmed IHC results. IHC was used to determine the relationship of P-Ser118-ER to known prognostic markers and active mitogen-activated protein kinase (MAPK; erk1/2) expression.
Results: P-Ser118-ER was significantly correlated with the expression of total ER, determined by ligand binding assay (r = 0.442, P = 0.002), but not with progesterone receptor expression or nodal status. P-Ser118-ER was inversely correlated with histological grade (r = -0.34, P = 0.023), reflecting a similar trend for total ER (r = -0.287, P = 0.056). Categorical contingency analysis confirmed that P-Ser118-ER was more frequently associated with lower than higher grade breast tumors (P = 0.038). In addition P-Ser118-ER was significantly associated with detection of active MAPK (Erk1/2; Spearman r = 0.649, P < 0.0001; Fisher’s exact test, P = 0.0004).
Conclusions: P-Ser118-ER detection is associated with a more differentiated phenotype and other markers of good prognosis in human breast cancer. P-Ser118-ER is correlated with active MAPK in human breast tumor biopsies, suggesting the possibility that active MAPK either directly or indirectly has a role in the regulation of P-Ser118-ER expression in vivo. These data provide evidence for a role of P-Ser118-ER in human breast cancer in vivo.
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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positive, and ER expression in breast tumors provides prognostic information (1)
, in addition to treatment response information (1)
. Targeting the ER using the selective ER modulator, tamoxifen, is efficacious in both the treatment and prevention of human breast cancer (2
, 3)
, and the basic rationale for tamoxifen therapy is that it binds directly to the ER, inducing conformational changes, such that estrogen signaling and therefore proliferation is inhibited in ER+ human breast cancer cells (4)
. However, despite tamoxifen therapy success, some primary ER-positive tumors do not respond to tamoxifen (de novo resistance), and many that originally respond to tamoxifen eventually acquire tamoxifen resistance, despite continued expression of ER (5
, 6)
.
ER is a ligand-activated transcription factor that can be phosphorylated at several sites (7)
. Phosphorylation of ER can be enhanced by estradiol binding and via activation of other signal transduction pathways (7)
. A well-studied phosphorylation site on ER is Ser118(7)
. Both estrogens and growth factors, such as epidermal growth factor (EGF), cause phosphorylation of Ser118-ER (7, 8, 9)
, and direct phosphorylation of Ser118-ER by mitogen-activated protein kinase (MAPK; Erk1/2) occurs in vitro (10)
. In addition, Ser118-ER is a major phosphorylation site resulting from activation of the ras-raf-MAPK (Erk1/2) pathway in vivo (8
, 11
, 12)
, and this process can be ligand independent (9
, 11)
. In contrast, it is unclear which kinase is responsible for the ligand i.e., estradiol, dependent phosphorylation of Ser118-ER, although Cdk7 is a candidate (9)
. Because ligand-independent ER activation may be a mechanism of altered estrogen and antiestrogen resistance in breast cancer, we undertook a study to determine whether P-Ser118-ER can be detected in human breast tissues, normal and neoplastic in vivo, and to determine any relationship to activated MAPK (Erk1/2).
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Human Breast Tumors.
All breast tumor cases used were selected from the Manitoba Breast Tumor Bank (Winnipeg, Manitoba, Canada). As described previously (13)
, tissues are accrued to the bank from cases at multiple centers within Manitoba, rapidly collected, and processed to create matched formalin-fixed, embedded, and frozen tissue blocks for each case with the mirror image surfaces oriented by colored inks. The histology of every sample in the bank is uniformly interpreted by a pathologist in H&E-stained sections from the face of the paraffin tissue block. This information is available in a computerized database, along with relevant pathological and clinical information, and was used as a guide for selection of specific paraffin and frozen blocks from cases for this study. For each case, interpretations included an estimate of the cellular composition, including the percentage of invasive epithelial tumor cells and stroma, tumor type, and tumor grade (Nottingham score). Steroid receptor status was determined for all primary tumor samples by ligand binding assay performed on an adjacent portion of tumor tissue. Tumors with ER levels >3 fmol/mg total protein were considered ER positive, and tumors with progesterone receptor (PR) levels >10 fmol/mg total protein were considered PR positive.
Cell Culture.
MCF-7 cells were routinely grown as described previously (14)
. For experiments, they were depleted of estrogens using medium containing phenol red-free DMEM and 5% twice charcoal-dextran-stripped fetal bovine serum, as described previously (15)
. The depleted cells were then left untreated or treated for 30 min with E2 (10 nM) plus human recombinant EGF (100 ng/ml) shown previously to increase the level of P-Ser118-ER (8)
. Proteins were extracted as described previously (15)
and subjected to Western blotting.
Western Blotting.
Extract aliquots were analyzed using 10% SDS-PAGE and 4% stacking gel at 200 V for 45 min at room temperature according to the Laemmli method (16)
. Gels were transferred to nitrocellulose for 1 h at 120 V at 4°C using 3-(cyclohexylamino)-1-propanesulfonic acid transfer buffer [10 mM 3-(cyclohexylamino)-1-propanesulfonic acid (pH 11), 20% methanol]. Blots were blocked overnight at 4°C in 0.2% (w/v) I-block (Tropix, Foster City, CA) in Tris-buffered saline (TBS) and incubated with a mouse monoclonal antibody specific for P-Ser118-ER (#2511, 16JR; Cell Signaling Technology, NEB Ltd., Mississauga, Ontario, Canada) at 1/1000 in 0.2% I-block in TBS containing 0.5% Tween 20 overnight at 4°C. This was followed by 5 x 5 min washes in TBST and then the secondary antibody for 1 h at room temperature (goat antimouse IgG- horseradish peroxidase, Jackson ImmunoResearch Labs, Inc., West Grove, PA; 1/5000 in TBST). The blot was washed 5 x 5 min with TBST and visualized using Supersignal West Dura Extended Duration Substrate kit (Pierce Chemical Co., Rockford, IL) according to the manufacturer’s instructions. Signal detection and documentation were using the ChemiDoc gel Documentation Systems 2000 (Bio-Rad Labs Canada, Mississauga, Ontario, Canada). The membrane was then stripped [0.2 M glycine, 0.1% SDS, and 1% Tween 20 (pH 2.2)] for 1 h at room temperature and blocked for 1 h at room temperature with 0.2% (w/v) I-block in TBS. The membrane was then probed with ER mouse monoclonal antibody (NCL-ER-6F11; Novocastra Labs Ltd., Newcastle-on-Tyne, England), 1/1000 in TBST buffer [20 mM Tris base, 0.137 M NaCl (pH 7.5), and 0.1% volume for volume Tween 20 containing 0.2% I-block], overnight at 4°C to determine total ER.
Immunohistochemistry (IHC).
IHC was performed on serial 5-µm sections from a representative formalin-fixed, paraffin-embedded archival tissue block from each tumor. In all cases, tissue samples were fixed for 18–24 h in 10% buffered formalin before routine embedding in paraffin wax. Sections were cut (5 µm), mounted on Superfrost/Plus slides (Fisherbrand), and dried overnight at 37°C. IHC for P-Ser118-ER used mouse monoclonal antibodies specific for ER phosphorylated on serine 118 (#2511, 16JR; Cell Signaling Technology, NEB Ltd.). Antibodies were applied using an automated tissue immunostainer (Discovery module; Ventana Medical Systems, Phoenix, AZ) and DAB IHC kit, and bulk reagents were supplied by the manufacturer. Briefly, the Discovery staining protocol was set to "Standard Cell Conditioning," followed by 12-h incubation with primary antibody and 32-min incubation with secondary antibody. Primary antibody concentrations initially applied to the Ventana instrument were 1:200 for P-Ser118-ER, translating into final concentrations of 1:600 after 1:3 dilution with buffer dispensed onto the slide with the primary antibody.
Phospho-p44/42 MAPK (Thr202/Tyr204) rabbit polyclonal, antibody (#9101S; NEB, Beverly, MA) specific for dually phosphorylated (active) forms of the MAPK isoforms, extracellular signal-regulated kinase (erk1 and 2, p44/42), has been validated previously for IHC. This antibody was used, as described previously (17)
. Briefly, sections were dewaxed in xylene (4 min) and rehydrated in graded alcohol. DAKO EnVision System, Peroxidase (DAKO Diagnostics Canada, Inc.) was used for IHC. Blocking steps included peroxidase blocking reagent (0.03% hydrogen peroxide containing sodium azide) for 5 min to block endogenous peroxidase and Universal Blocker (DAKO Diagnostics Canada, Inc.) for 15 min to prevent nonspecific staining with antibody from both mouse and rabbit. Tissue sections were incubated overnight at 4°C with primary antibody (1:250 in Antibody Diluting Buffer; DAKO Diagnostics Canada, Inc.) after an initial incubation, with the same antibody at 37°C for 30 min. After overnight incubation, slides were treated with labeled polymer (goat antirabbit immunoglobulin in Tris-HCl buffer containing carrier protein and antimicrobial agent) for 30 min at room temperature. Finally, slides were incubated for 10 min with Substrate AEC Chromogen (3-amino-9-ethylcarbazole). Each incubation was followed by 2 x 2 min TBS washes. The slides were counterstained with hematoxylin, immersed in a bath of ammonia water, and rinsed in distilled water, and a coverslip was applied using aqueous mounting medium. Levels of nuclear P-Ser118-ER and active MAPK expression were scored semiquantitatively, under the light microscope. Scores were obtained by estimating average signal intensity (scale of 0–3) and the proportion of epithelial cells showing a positive signal (0–100%). The intensity and proportion scores were then multiplied to give an overall IHC score.
Statistical Methods.
Correlation between P-Ser118-ER protein level (IHC score) and tumor characteristics was tested by calculation of the Spearman coefficient r. Fisher’s exact test was used to test for differences in frequency of detection of P-Ser118-ER (positive tumors were defined as sections containing any detectable nuclear staining) between higher and lower grade tumors (lower grade was defined as score 
6, and higher grade was defined as scores 
7). In addition, Fisher’s exact test was used to test for differences in frequency of detection of P-Ser118-ER as a binary factor, using the previous definition, between tumors positive and negative for active MAPK expression (positive tumors were defined as containing any cells with detectable staining).
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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in Human Breast Tumors in Vivo. in human breast tumors in vivo was investigated using IHC and an antibody specific for P-Ser118-ER, as described in "Materials and Methods." This antibody was shown previously to detect P-Ser-ER immunocytochemically (18)
. However, the ability of the antibody to detect P-Ser118-ER by IHC in formalin-fixed, paraffin-embedded human breast biopsy samples was not validated previously to our knowledge. To do this estrogen, depleted MCF-7 cells were treated for 3 h with 50 µM mitogen-activated protein/extracellular signal-regulated kinase kinase inhibitor, PD98059, or vehicle (DMSO) alone. An aliquot of the cells from each group was extracted and analyzed by Western blotting. The remainder of the cells was embedded in 3% agar, formalin fixed, paraffin embedded, and processed for IHC (19)
. Western blot analysis (Fig. 1)
showed a decrease in P-Ser118-ER in the PD98059-treated cell extracts compared with the vehicle alone-treated cells. The positive and negative controls were extracts of estrogen-depleted MCF-7 cells treated with 10 nM E2 plus 100 ng/ml human recombinant EGF for 30 min or treated with vehicle alone, respectively (8)
. Changes seen in P-Ser118-ER were not paralleled in total ER levels when the blot was stripped and reprobed with an antibody for total ER, supporting the conclusion that inhibition of mitogen-activated protein/extracellular signal-regulated kinase kinase decreased active MAPK (Erk1/2, p42/44; Ref. 17
), which directly or indirectly reduced expression of P-Ser118-ER. IHC using P-Ser118-ER-specific antibody showed presence of nuclear staining in some but not all cells in the DMSO-treated cells, but no detectable staining was present in PD98059-treated cells (Fig. 2A
, PD treated and 2B, vehicle alone). This result paralleled that obtained by Western blotting.
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was obtained in 20 (44.4%) tumors. Examples of P-Ser118-ER IHC in breast tumor sections are shown in Fig. 2, C
(negative for P-Ser118-ER) and D (positive for P-Ser118-ER). Somewhat <50% of tumor sections contained adjacent normal tissue in the same section, and no detectable staining for P-Ser118-ER was seen in any of the normal breast tissue (data not shown), suggesting that expression of P-Ser118-ER is increased in breast tumors compared with normal breast tissue. Western blotting of human breast tumor extracts also demonstrated the presence of P-Ser118-ER in extracts of some tumors (Fig. 3)
.
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in Human Breast Tumors with Known Prognostic Markers. expression with PR expression, tumor size, or nodal status. However, as might be expected, a positive correlation of P-Ser118-ER was seen with ER expression, measured by ligand binding assay (r = 0.442, P = 0.002), although three tumors classified as ER- by ligand binding, had detectable P-Ser118-ER. This may be because of tumor heterogeneity, a false negative ligand binding assay attributable to technical issues, or the possibility that ER present in these tumors was not functional with respect to ligand binding activity. Interestingly, an inverse relationship of P-Ser118-ER IHC score with grade (r = -0.34, P = 0.023) was observed. The median IHC score of P-Ser118-ER was significantly higher (Mann-Whitney two-sided, P = 0.047) in the lower grade tumors (6) than higher grade tumors (7). There was also a trend to an inverse association of total ER with grade (r = -0.287, P = 0.056), a result consistent with the correlation of P-Ser118-ER with total ER. In the same tumor samples, as expected, total ER expression as measured by ligand binding was significantly correlated with PR expression measured by ligand binding (r = 0.43, P = 0.003).
Fisher’s exact test was used to examine further the inverse relationship of P-Ser118-ER with grade and test for differences in the frequency of detection of P-Ser118-ER (positive or negative) between higher and lower grade tumors. This analysis showed that P-Ser118-ER was more frequently associated with lower than higher grade breast tumors, and lack of detectable P-Ser118-ER was more frequently associated with higher grade tumors (P = 0.038).
Correlation of P-Serine-118-ER in Human Breast Tumors with Active MAPK (Erk1/2, p42/44).
Because active MAPK (Erk1/2, p42/44) can directly phosphorylate ER on Ser118 and the activation of the ras-raf-MAPK pathway has been implicated in phosphorylation of ER on Ser118in vivo, we were interested to determine whether this pathway may be involved in the generation of P-Ser118-ER in vivo. Therefore, we measured active MAPK (erk1/2, p42/44) expression by IHC score in adjacent sections from the same tumors in which we had P-Ser118-ER determined previously. An example of the data obtained is shown in Fig. 2, E
(negative) and F (positive). Spearman analysis showed a significant positive correlation of active MAPK IHC score and P-Ser118-ER IHC score (r = 0.649, P < 0.0001). Fisher’s exact test was used to examine further the relationship of P-Ser118-ER with active MAPK expression and test for differences in the frequency of detection of P-Ser118-ER (positive or negative) between tumors with positive active MAPK (any detectable IHC) and negative active MAPK. This analysis showed that P-Ser118-ER was associated with active MAPK expression, and lack of detection of P-Ser118-ER was associated with lack of detection of active MAPK (P = 0.0004, two tailed).
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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has only recently been shown by Western blotting (9)
, and our current data confirm this observation. This provides evidence of a role for P-Ser118-ER in human breast tumors in vivo. Therefore, we have investigated further P-Ser118-ER expression in human breast tumors in vivo, using IHC, and this is the first time to our knowledge that the expression of P-Ser118-ER has been investigated in multiple human breast tissue biopsy samples. We validated the use of a commercially available antibody specific for P-Ser118-ER for IHC in formalin-fixed, paraffin-embedded breast tumor biopsy samples and detected specific, nuclear staining for P-Ser118-ER in 
45% of the breast tumors analyzed. As would be expected, the frequency and expression level of P-Ser118-ER were positively correlated with the level of ER expression determined previously by ligand binding assays on the same samples. In those tumor sections in which adjacent normal breast tissue was also present, there was little if any detection of P-Ser118-ER which suggests that an up-regulation of P-Ser118-ER expression occurs in breast tumors compared with normal breast tissue. This again probably reflects the correlation of P-Ser118-ER with total ER expression, because ER expression is also known to be up-regulated in ER+ breast tumors compared with normal breast tissue (20
, 21)
. However, any change in the relative expression of P-Ser118-ER to total ER in normal breast versus breast cancer remains unknown, because of sensitivity issues in measuring even total ER by Western blot in normal human breast tissue. Although total ER in this cohort of tumors was found to correlate with PR expression, a known estrogen-regulated gene product, surprisingly no such correlation of P-Ser118-ER with PR levels was found, suggesting that regulation of ER activity via phosphorylation of Ser118 may not be involved in the mechanism by which estrogens regulate PR expression. Because the mechanisms, beyond ER, by which estrogen regulates PR expression, are distinct from those by which estrogen induces growth stimulation in breast cancer cells as underscored by their dissociation in some cell line models (22)
, it is tempting to speculate that P-Ser118-ER may be involved in and/or a marker of an intact estrogen growth responsive pathway and therefore may more accurately predict sensitivity to hormonal therapies, all of which are aimed at inhibiting the growth stimulation effect of estrogen on breast cancer cells.
Interestingly, an inverse correlation of P-Ser118-ER with tumor grade was found, and a similar trend with total ER status was also found, although this did not reach statistical significance. Histological grade in breast cancer has been demonstrated several times to show a strong correlation with prognosis (23
, 24)
. Histological grade using the Nottingham method integrates scores from glandular differentiation, nuclear morphology, and mitotic counts (23
, 24)
. Higher grade is significantly associated with poor outcome and survival. Furthermore, grade has been shown previously to correlate well with ER status (25)
, the possible biological explanation being that presence of ER expression is a biomarker of a more differentiated phenotype with a lower proliferative capacity. In addition to ER+, lower grade tumors are thought to be more likely dependent on the estrogen-regulated ER signal transduction pathway and therefore more likely to respond to endocrine therapy, consistent with their better outcome. Our data suggest that the detection of P-Ser118-ER may more accurately determine a ligand-dependent ER signal transduction pathway within a breast tumor, corresponding to a more differentiated phenotype.
Therefore, it was somewhat surprising that in the same breast tumor cohort, we found a significant positive association of active MAPK with the detection of P-Ser118-ER. This could be interpreted to mean that active MAPK directly or indirectly is responsible for the phosphorylation of ER on Ser118 in breast tumors in vivo. But this does not support the idea that P-Ser118-ER is a marker of ligand-independent activity of ER and therefore a poor prognostic factor. However, in cells in culture, it is E2-induced phosphorylation of Ser118 that results in highest levels of Ser118 phosphorylation, which is sustained for a longer period of time, compared with that induced by growth factor activation of the MAPK pathway (7
, 8)
. In addition, it has been suggested that active MAPK is involved in sensitizing cells to E2 and associated with hypersensitivity to E2, in the absence of directly phosphorylating ER at Ser118 (18
, 26
, 27)
. Such activation of ER is still responsive to antiestrogen inhibition (18)
, as opposed to the acquisition of ligand independence of the ER signal transduction pathway, possibly attributable to or in combination with chronic growth factor receptor activation of the MAPK and other pathways, resulting in permanent and antiestrogen resistant activation of ER, which in vivo may also be associated with reduced levels of ER expression, a finding consistent with cell line models where chronic, overexpression of EGF-R or HER-2 and hyperactivity of MAPK, at least, is associated with down-regulation of and/or loss of ER expression (28)
.
In conclusion, we have demonstrated the presence of ER specifically phosphorylated at Ser118 in human breast cancer biopsy samples by IHC, providing evidence of a role in human breast cancer. The relationship of P-Ser118-ER expression with known biomarkers in breast tumors suggests that P-Ser118-ER detection is associated with a more differentiated phenotype and other markers of good prognosis in human breast cancer. Whether it proves to be more accurate than the measurement of total ER in this regard, as well as its value in prediction of treatment response, remains to be determined from survival and outcome studies. In addition, the correlation of P-Ser118-ER with active MAPK (Erk1/2) suggests the possibility that active MAPK either directly or indirectly has a role in the regulation of P-Ser118-ER expression in vivo.
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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.
Note: Present address of Adewale Adeyinka: Division of Laboratory Genetics, Department of Laboratory, Medicine and Pathology, Mayo Clinic.
Requests for reprints: Leigh Murphy, Manitoba Institute of Cell Biology, Departments of Biochemistry & Medical Genetics, University of Manitoba, 675 McDermot Avenue, Winnipeg, Manitoba, Canada. Phone: (204) 787-4071; Fax: (204) 787-2190; E-mail: lcmurph@cc.umanitoba.ca.
Received 8/28/03; revised 10/21/03; accepted 11/10/03.
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REFERENCES |
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at serine 118 by two distinct signal transduction pathways revealed by phosphorylation-specific antisera. Oncogene, 21: 4921-4931, 2002.[CrossRef][Medline]
and ß mRNA expression during human breast tumorigenesis. Cancer Res., 58: 3197-3201, 1998. expression in breast cancer cells. Mol. Endocrinol., 15: 1344-1359, 2001.This article has been cited by other articles:
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