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. 2008 Dec 24;135(7):933–941. doi: 10.1007/s00432-008-0529-5

Phospho-mTOR and phospho-4EBP1 in endometrial adenocarcinoma: association with stage and grade in vivo and link with response to rapamycin treatment in vitro

Silvia Darb-Esfahani 1,✉, Areeg Faggad 1, Aurelia Noske 1, Wilko Weichert 1, Ann-Christin Buckendahl 1, Berit Müller 1, Jan Budczies 1, Annika Röske 1, Manfred Dietel 1, Carsten Denkert 1
PMCID: PMC12160191  PMID: 19107520

Abstract

Purpose

Endometrial adenocarcinoma, due to a frequent activation of PI3 K/AKT has been proposed as a candidate neoplasm for the treatment with mTOR inhibitors. Yet, data on the expression of mTOR cascade components in endometrial cancer are lacking.

Methods

To provide a basis for futher studies with mTOR inhibitors, we used immunohistochemistry to evaluate the expression of activated mTOR pathway components in 57 endometrial cancer surgical specimens in vivo, and investigated in vitro the relation between the activation of AKT/mTOR and the response to rapamycin.

Results

p-mTOR expression was associated with nuclear p-4EBP1 expression (P = 0.02), and was more frequent in tumors extending ouside the uterine corpus (P = 0.011). Nuclear p-4EBP1 expression was increased in carcinomas of poor differentiation (P = 0.012). In cultivated PTEN-deficient Ishikawa cells, in addition to an activation of AKT, a phosphorylation of mTOR and 4EBP1 was evident, while PTEN-wild type HEC-1A cells lacked AKT activation but revealed a reduced expression of p-mTOR and p-4EBP1. Rapamycin induced a growth reduction, which was clearly more pronounced in Ishikawa cells than in HEC-1A cells (P < 0.03) and could be observed for up to 6 days.

Conclusisons

Expression of mTOR and 4EBP1 characterize high-grade, high-stage endometrial adenocarcinomas and might be predictive markers of a response to rapamycin. Based on our results, we suggest that the expression of elements of the mTOR pathway in human tumor tissue should be further evaluated as a possible predictive marker in large-scale clinical studies as well as translational research protocols in clinical studies with mTOR inhibitors.

Keywords: PI3 K/AKT/mTOR pathway, Rapamycin, Endometrial adenocarcinoma

Introduction

Endometrial adenocarcinoma is the most frequent malignancy of the female genital tract. A total of 39,080 new cases leading to 7,400 cancer-related deaths have been expected for the year 2007 in the US (Jemal et al. 2007). More than 80% of the uterine tumors are endometrioid adenocarcinomas, which together with the very rare mucinous subtype are summarized as type 1 tumors. These type 1 tumors, in contrast to the very aggressive serous or clear cell endometrial adenocarcinomas (type 2 tumors), arise in the context of unopposed estrogen exposure (Prat 2004).

Overall prognosis is relatively good, because the predominant histologic subtype, endometrioid adenocarcinoma, is commonly diagnosed at early stage, when tumor growth is still confined to the uterus and the patient can be cured by hysterectomy. However, for patients with tumors extending outside the uterine corpus and tumors of low differentiation and type 2 histology, survival rates decline sharply. Disease recurrence and disease-related death is common in this patient group. While in FIGO stage I tumors the 5-year survival rate is more than 90%, in stage II carcinomas the 5-year survival rate drops to 70% and to 60% in stage III tumors (Steiner et al. 2003). Similarly, while patients with grade 1 tumors achieve 5-year survival rates of more than 90%, in patients with grade 2 and 3 tumors survival rates are decreased to 80 and 50%, respectively (Steiner et al. 2003). Current options for these patients are radiotherapy and cytotoxic therapy, in addition to surgery. Obviously, new therapeutic options are thus needed in particular for patients with high-risk endometrial cancer.

The signal transduction pathway comprising the kinase mTOR (mammalian target of rapamycin) is one of the most promising therapeutic targets in cancer research. The mTOR protein forms a complex with adaptor proteins, mTORC1 and mTORC2 (mTOR complex) (Sabatini 2006). Activation of mTORC1 occurs in response to growth factors and nutrients, and mTORC1 activation itself regulates cell growth by modulating protein synthesis, ribosome biogenesis and autophagy (Wullschleger et al. 2006). Many cancer-promoting processes activate the mTOR pathway. The role of mTORC2 is less clearly defined yet. Activated mTORC1 phosphorylates a couple of downstream effectors, e.g., eukaryotic translation initiation factor 4E binding protein 1 (4EBP1). Hypophosphorylated 4EBP1 binds to and thereby inactivates the cap-binding protein eukaryotic translation initiation factor 4E (eiF4E), while after phosphorylation by mTOR 4EBP1 releases eiF4E and allows its binding to the cap-structure of mRNA and the beginning of protein translation (Sabatini 2006). Especially, 4EBP1 phosphorylation at Thr70 has been reported to correlate very well with the activity of the mTOR pathway (Dudkin et al. 2001). Rapamycin is a natural macrolid, which specifically inhibits mTOR by disturbing the formation of the mTORC1, while mTORC2 seems to be less sensitive to the effects of rapamycin (Sabatini 2006).

A key negative regulator of mTORC1 is the TSC1 (tuberous sclerosis 1)–TCS2 (tuberin) complex, which is inhibited by the well-characterized oncogenic molecule AKT (protein kinase B, PKB), thereby activating mTOR (Sabatini 2006). In addition, AKT has been reported to be involved in the phosphorylation and activation of mTOR at Ser2448 (Nave et al. 1999). AKT thereby tightly interconnects the mTOR cascade with the phosphatidylinositol 3-kinase (PI3 K) pathway, which itself is essentially regulated by the phosphatase and tensin homolog deleted on chromosome 10 protein (PTEN) (Chow and Baker 2006). PTEN, a lipid phosphatase, dephosphorylates phosphatidylinositol (3,4,5) triphosphate (PIP3), the product of PI3 K, to phosphatidylinositol (4,5) bisphosphate, resulting in a reduced phosphorylation of AKT by 3-phosphoinositide-dependent protein kinase-1 (PDK-1) at Ser473 (Bayascas and Alessi 2005). PTEN loss due to mutational alterations or epigenetic silencing is frequently found in tumors of the ovary, breast or prostate (Chow and Baker 2006). In all those cancers PTEN loss occurs relatively late, but in endometrioid endometrial adenocarcinoma PTEN is already found in 20% of the pre-invasive precursor lesions, the atypical endometrial hyperplasias, and in invasive cancers the rate of PTEN loss is 50–80% (Chow and Baker 2006; Hecht and Mutter 2006). PTEN loss is associated with high levels of AKT activation in endometrial adenocarcinoma (Kanamori et al. 2001), which then leads to the activation of survival signals by phosphorylation of several targets, such as BAD, glycogen synthase kinase-3 (GSK-3), forkhead transcription factor (FKHR) and caspase-9 (Chow and Baker 2006). However, there are further mechanisms of AKT activation in endometrial adenocarcinoma, such as mutations of the PI3 K gene (Oda et al. 2005).

Due to the overactivity of AKT, PTEN-deficient cancers are thought to respond well to inhibitors of the mTOR pathway, like rapamycin or its synthetic analogues (Rowinsky 2004). Several clinical trials have already been performed or are ongoing for testing these substances in endometrial adenocarcinoma (Easton and Houghton 2006; Pectasides et al. 2007; Gadducci et al. 2006) and in other tumors. Although high AKT activity is a known feature of endometrial adenocarcinoma, very little data exist on the role of the mTOR pathway in this cancer and data on the expression of mTOR cascade components in vivo are lacking. Furthermore, few publications exist reporting the dependency of rapamycin response to the expression of biomarkers like PTEN or PI3 K/AKT/mTOR pathway components in endometrial cancer.

In this project, we evaluated whether an activation of the mTOR pathway might be detectable in endometrial cancer tissue to potentially define biomarkers that might serve to select patients who may profit from a therapy with mTOR inhibitors. In vitro, we further investigated the relation between the activation of AKT/mTOR and the response to rapamycin treatment.

For this purpose, we performed immunohistochemical staining of mTOR cascade components in endometrial adenocarcinoma surgical specimens, and investigated the proliferation of endometrial cancer cells after treatment with rapamycin.

Patients, materials and methods

Study population

Our study group comprised 57 patients who underwent surgery for primary endometrial cancer in the Department of Gynecology and Obstetrics of the Charité University Hospital from 1988 to 2004. Median age at surgery was 65 years, range: 50–88 years. Hysterectomy specimens of all patients were examined in the Institute of Pathology, Charité University Hospital. Tumor histology and grading according to WHO (2003) (Tavassoli and Devilee 2003) were controlled by an experienced gynecopathologist (C.D.) for all cases. Tumor histology was endometrioid in 50 cases (87.6%) and mucinous in 1 case (1.8%). Four tumors were serous (7.0%), one was a clear cell carcinoma (1.8%) and one a mixed serous and clear cell adenocarcinoma (1.8%). The majority of the tumors were early stage cancers (pT1: 42 cases, 73.7%; pT2: 7 cases, 12.3%; pT3: 8 cases, 14%). Lymphadenectomy was performed in 20 patients (35.1%) with 3 patients being nodal positive (15.0%). Most tumors were well differentiated (G1: 31 cases, 54.4%; G2: 13 cases, 22.8%, G3: 13 cases, 22.8%). This study was approved by the ethics committee of the Charité Hospital.

Immunohistochemical staining

Immunohistochemical examination was performed on tissue microarrays. For this purpose, representative tumor areas were marked on the routine H&E stained histological sections. Four tissue cores of 1.5 mm diameter from representative tumor areas of the donor blocks were punched using a tissue micro-arrayer (Beecher Instruments, Woodland, USA) and positioned in a recipient paraffin array block. Immunohistochemistry was performed according to standard procedures. Briefly after deparaffinization, the slides were boiled in 0.01 M sodium citrate buffer (pH 6.0) in a pressure cooker for 5 min. Two different protocols turned out to provide optimal staining results for p-4EBP1 and p-mTOR. For the detection of p-4EBP1, the slides were incubated with a rabbit polyclonal antibody for detecting 4EBP1 protein phosphorylated at Thr70, dilution 1:25 (Cell Signaling, Danvers MA Technology, USA) for 1 h at room temperature. Immunostaining was followed by incubation with EnVision detection system (DAKO, Glostrup, Denmark) and visualized using DAB (diaminobenzidine) chromogen solution (DAKO). p-mTOR was detected using an IHC (immunohistochemistry)-specific rabbit monoclonal antibody for detecting mTOR protein phosphorylated at Ser2448 (clone 49F0, Cell Signaling Technology). The slides were incubated with primary antibody diluted 1:50 in antibody diluent solution (Zymed, San Francisco, CA, USA) for 20 min at room temperature and then at 4°C overnight. After washing slides in TBS, a streptavidin-biotin system was applied according to a standard protocol as provided by the manufacturer (BioGenex, San Ramon, CA, USA). For color development, a fast red system (Sigma, Deisenhofen, Germany) was used. After color development was stopped, the slides were cover slipped with Aquatex (Merck, Gernsheim, Germany).

Interpretation of immunohistochemical staining

Immunohistochemical staining was evaluated by a semiquantitative scoring system. Both cytoplasmic and nuclear stainings were evaluated separately. Staining intensity was scored as 0 = negative, 1 = weak, 2 = moderate, 3 = strong. The percentage of cells stained were scored as 0 = no cells stained, 1 = 1–10% of cells stained, 2 = 11–50% of cells stained, 3 = 51–80% of cells stained, 4 = more than 80% of cells stained. The two parameters were multiplied, resulting in an individual immunoreactivity score (IRS) ranging from 0 to 12 for every case. To separate cases with high expession of p-4EBP1 from cases with low-level expression, we considered cases with an IRS > 4 as “positive”, and cases with an IRS of 0–4 as “negative”. As p-mTOR was only weakly and focally expressed, we used a lower cutoff point for separating cases with “negative” (IRS 0-3) and “positive” expression (IRS > 3).

Cell lines

The human endometrial adenocarcinoma cell line HEC-1A was obtained from the American Type Culture Collection (ATCC, Rockville, MD), and the human endometrial adenocarcinoma cell line Ishikawa was obtained from the European Collection of Cell Cultures (ECACC, Salisbury, UK). Cell lines were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. Cells were maintained at 37°C in a humidified atmosphere of 5% CO2/95% air. Rapamycin (Cell Signaling) was added in different concentrations (20–1,000 nM) for up to 6 days. A change of medium with a subsequent replacement of repaying was performed after 3 days of incubation. The inhibitor was dissolved in DMSO. All cell culture experiments were performed at least in triplicate.

Western blot

For protein analysis, the cells were lysed in 100 μl of 62.5 mM Tris–HCl (pH 6.8) containing 2% sodium dodecyl sulfate, 10% glycerol, 50 mM DTT and 0.1% bromophenole blue. A weight of 100 μg protein/sample was separated on a 10% polyacrylamide gel, blotted onto nitrocellulose membranes (Schleicher&Schuell, Dassel, Germany), washed in PBS and blocked in buffer (1× PBS, 0.1% Tween-20, 5% I-block (Tropix, Bedford, MA, USA) for 1 h at room temperature. The membranes were probed with antibody overnight at 4°C and diluted 1:1,000 in blocking buffer, followed by incubating with alkaline phosphatase-conjugated goat-anti-rabbit secondary antibody (Tropix). The bands were visualized using the CDP star RTU luminescence system (Tropix). Antibody concentrations were 1:1,000 for the anti-p-mTOR antibody (ProSci, Poway CA, USA), anti-total-4EBP1 antibody (Cell Signaling), anti-p-4EBP1 antibody (Cell Signaling), anti-total-AKT antibody (1:1,000), anti-p-AKT antibody (1:1,000), as well as for the anti-PTEN antibody (1:1,000), and 1:3,000 for the monoclonal anti-actin antibody (Chemicon, Temecula, USA).

The XTT assay

The cells were incubated in 96-well plates (3,000 cells/well) with or without rapamycin. Cell proliferation was determined by using an XTT-based colorimetric assay (Roche Diagnostics, Mannheim, Germany) according to the manufacturer’s instructions. Measurements were performed 3 h after addition of the XTT reagent.

Statistical evaluation

Immunohistochemical expression data were correlated with clinicopathological data by the use of the Fisher’s exact test. Differences in cell proliferation rates as measured by XTT assay were assessed by two-sided columnar t-test or by two-sided one-sample t-test, as indicated. P values smaller than 0.05 were considered to be significant. For statistical procedures, the software packages SPSSv15.0 and GraphPad Prism 4.0 were used.

Results

The mTOR cascade is activated in a subset of endometrial adenocarcinomas

To investigate whether an activation of the mTOR pathway could be detected in surgical specimens of endometrial cancer, we used immunohistochemistry to identify the activated forms of mTOR (p-Ser2488) and its downstream effector 4EBP1 (p-Thr70) in human tumor tissue. Staining data for mTOR were available for 56 patients. Phosphorylated mTOR typically displayed a patchy immunostaining in endometrial adenocarcinoma specimens (Fig. 1a, b). When present, the staining intensity was weak to moderate and a large fraction of cases did not show any staining (immunoreactivity score 0, 19 cases, 33.9%).

Fig. 1.

Fig. 1

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Immunohistochemical detection of p-mTOR and p-4EBP1 in endometrioid adenocarcinoma specimens: a moderate, focal p-mTOR expression is evident in a well-differentiated adenocarcinoma (200×) (a). Strong p-mTOR expression in a G3 endometriod adenocarcinoma. An increase in staining intensity from adenoid tumor structures (on the left) to solid tumor areas (on the right) is visible (400×) (b). Strong cytoplasmic and concomitant moderate nuclear p-4EBP1 expression is seen in a well-differentiated adenocarcinoma (200×) (c). Focally accentuated, strong nuclear p-4EBP1 expression in the presence of weak cytoplasmic expression is evident in infiltrating glands of a moderately differentiated adenocarcinoma (200×) (d)

Staining data for 4EBP1were available for 54 patients. In most cases, p-4EBP1 was diffusely expressed in the cytoplasm of neoplastic epithelial cells showing weak to moderate intensity (Fig. 1c, d). Most cases also displayed a distinct nuclear p-4EBP1 expression of varying intensity. p-mTOR was significantly associated with nuclear p-4EBP1 expression (P = 0.02, data not shown). There was a trend towards the association of nuclear and cytoplasmic p-4EBP1 expression (P = 0.098, data not shown). Cytoplasmic p-4EBP1 and p-mTOR expression was not related to each other (P > 0.05, data not shown).

Expression of p-mTOR and phospho-4EBP1 is increased in high-risk tumors

Endometrial adenocarcinomas can be characterized by a variety of clinical and pathological parameters, which help to estimate cancer behavior. To evaluate whether an activation of the mTOR cascade might be linked to certain tumor properties, we studied the distribution of p-mTOR and p-4EBP1 expression between different clinicopathological categories. The positivity of p-mTOR was significantly more frequent in tumors extending outside the uterine corpus (pT2 and pT3, P = 0.011, Table 1) and accordingly, tended to increase in advanced stage tumors (FIGO II–III, P = 0.055, Table 1). There was further a trend towards a higher p-mTOR expression in cases with lymph node metastasis (P = 0.088, Table 1). Nuclear p-4EBP1 expression was significantly increased in carcinomas of poorer differentiation (G2-3, P = 0.012), and tended to be more frequent in tumors with deep infiltration of the myometrium (pT1a + b vs. pT1c, P = 0.05). Cytoplasmic p-4EBP1 expression was not related to clinicopathological characteristics (Table 1). Similar associations between p-mTOR as well as p-4EBP1 expression were evident in the subgroup of type 1 tumors (data not shown).

Table 1.

Associations of p-mTOR and p-4EBP1 expression with clinicopathological factors

Characteristic All cases
n = 54		 p-4EBP1
cytoplasmic
positive
n = 23		 P p-4EBP1
nuclear
positive
n = 23		 P All cases
n = 56		 p-mTOR
positive
n = 18		 P
Age at surgery
 ≤60 years 19 (35.2%) 11 (20.4%) 0.149 7 (13.0%) 0.577 20 (35.7%) 6 (10.7%) 1.000
 >60 years 35 (64.8%) 12 (22.2%) 16 (29.6%) 36 (64.3%) 12 (21.4%)
Histologic type
 Type 1 48 (88.9%) 20 (37.0%) 1.000 20 (37.0%) 1.000 50 (89.3%) 17 (30.4%) 0.652
 Type 2 6 (11.1%) 3 (55.6%) 3 (55.5%) 6 (10.7%) 1 (1.8%)
pT
 pT1 40 (74.1%) 20 (37.0%) 0.115 15 (27.8%) 0.226 41 (73.2%) 9 (16.1%) 0.011
 pT2-3 14 (25.9%) 3 (55.5%) 8 (14.8%) 15 (26.8%) 9 (16.1%)
Myometrial infiltration n = 40 n = 41
 pT1a + b 22 (55.0%) 12 (30.0%) 0.751 5 (12.5%) 0.050 24 (58.5%) 5 (12.2%) 1.000
 pT1c 18 (45.0%) 8 (20.0%) 10 (25.0%) 17 (41.5%) 4 (9.8%)
pN n = 19 n = 20
 pN0 16 (84.2%) 8 (42.1%) 0.228 7 (36.8%) 0.582 17 (85.0%) 2 (10.0%) 0.088
 pN1 3 (15.8%) 0 (0.0%) 2 (10.5%) 3 (15.0%) 2 (10.0%)
FIGO stage
 FIGO I 40 (74.1%) 20 (37.0%) 0.115 16 (29.6%) 0.546 41 (73.2%) 10 (17.9%) 0.055
 FIGO II–III 14 (25.9%) 3 (55.5%) 7 (13.0%) 15 (26.8%) 8 (14.3%)
Grading
 G1 28 (51.9%) 14 (25.9%) 0.271 7 (13.0%) 0.012 30 (53.6%) 7 (12.5%) 0.159
 G2–3 26 (48.1%) 9 (16.7%) 16 (29.6%) 26 (46.4%) 11 (19.6%)
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Percentages for all cases are indicated in brackets

P-value, Fisher′s exact test

In vitro, the AKT/mTOR pathway is activated in Ishikawa endometrial cancer cells and is inhibited by rapamycin treatment

In order to examine the interconnection between the PTEN state, AKT activation and the mTOR pathway in vitro, we performed Western blot analysis of PTEN wild type HEC-1A and PTEN-deficient Ishikawa endometrial cancer cells. As expected, Ishikawa cells lacking the PTEN protein showed strong AKT phophorylation in contrast to HEC-1A cells (Fig. 2). Additionally, a phosphorylation of mTOR and 4EBP1 was evident in Ishikawa cells, indicating an activation of the mTOR cascade in these cells. In contrast, in HEC-1A cells, a smaller amount of phosphorylated mTOR as well as 4EBP1 was detectable too.

Fig. 2.

Fig. 2

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Western blot analysis of HEC-1A and Ishikawa endometrial cancer cells: cells were incubated with increasing concentrations of rapamycin for 48 h. Control: untreated cells, incubated in DMSO

Rapamycin treatment resulted in a decline of 4EBP1 phosphorylation in both cell lines, indicating a successful mTOR inhibition even with the lowest dose of 20 nM rapamycin (Fig. 2). The mTOR phosphorylation was also decreased by rapamycin treatment. Interestingly, the amount of total 4EBP1 protein also declined. Rapamycin treatment resulted in a slight decrease of AKT protein levels in both cell lines, yet did not lead to increased AKT activation in Ishikawa cells as the level of phosphorylated AKT remained stable throughout the experiment.

Rapamycin strongly inhibits the growth of Ishikawa cells and to a lesser extent the growth of HEC-1A cells

We then evaluated whether an inhibition of the mTOR pathway had an impact on cell growth, using XTT cell proliferation assay. As shown in Fig. 3a, rapamycin reduced the growth of Ishikawa cells significantly, 53–43% after 2 days of incubation, an effect that was much weaker in HEC-1A cells (comparison HEC-1A vs. Ishikawa for each concentration of rapamycin: P < 0.03, t-test). Here, a significant, yet small, growth reduction to 76–84% could be observed. Of note, a certain weak dose-dependency of the growth reduction was visible, but a strong effect was already achieved with a rather low rapamycin concentration (20 nM). To further evaluate the long-term effect of rapamycin treatment, we incubated the cells with 100 nM rapamycin for 3 and 6 days, respectively (Fig. 3b). The effect of rapamycin on cell proliferation at these later time points was similar to the earlier effect. In HEC-1A cells, a growth reduction to 83% was seen after 3 days and to 77% after 6 days. In Ishikawa cells, growth was reduced to 62% after 3 days and to 53% after 6 days of incubation. Again, the growth reduction was significantly stronger in Ishikawa than in HEC-1A cells (P < 0.003, t-test).

Fig. 3.

Fig. 3

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XTT assay of HEC-1A and Ishikawa cells incubated with varying concentrations of rapamycin for 2 days (a). XTT assay of HEC-1A and Ishikawa cells incubated with 100 nM rapamycin for 3 and 6 days, respectively (b). Dark gray boxes represent the data of HEC-1A cells, and light gray boxes represent data of Ishikawa cells. Asterisks indicate significant P-values and refer to the comparison between cells incubated with DMSO only and cells treated with rapamycin (columnar t-test); asterisks in connection with brackets refer to the comparison between HEC-1A and Ishikawa cells (t-test); whiskers indicate standard deviations. *P < 0.03; **P < 0.003; control: untreated cells, incubated in DMSO

Discussion

Our study, to our knowledge, for the first time demonstrates an activation of the mTORC1 pathway in a subset of endometrial cancers in vivo. This activation was more prevalent in high stage, high grade carcinomas. Furthermore, we found in vitro that in HEC-1A and Ishikawa endometrial carcinoma cells, a PTEN-deficiency and AKT activation as well as higher levels of mTOR and 4EBP1 phosphorylation predict a stronger response to rapamycin treatment as compared to PTEN wild-type cells.

In line with our findings of an association of mTOR pathway components with aggressive tumor features, several groups have investigated the expression pattern of mTOR and its downstream targets in other types of human cancers, thus providing additional translational evidence of an activation of the mTOR pathway in the malignant phenotype. An overexpression of total or phosphorylated 4EBP1 was found in carcinomas of the prostate (Kremer et al. 2006) and the breast (Zhou et al. 2004). Castellvi et al. (2006) reported an independent prognostic impact of the expression of the 4EBP1 protein, phosphorylated at Thr70, in ovarian cancer and the same group found that this marker was increased in the subset of poorly differentiated, high stage breast carcinomas with frequent locoregional recurrence (Rojo et al. 2007). Based on the findings of their group and of others, the authors suggest p-4EBP1 as a “funnel factor” on which several oncogenic pathways converge and which may be an important biomarker for therapy and prognosis prediction (Armengol et al. 2007); phospho-4EBP1 as well as phospho mTOR expression were further independent prognosticators for survival of breast cancer and biliary tract carcinoma patients (Castellvi et al. 2006; Herberger et al. 2007). The repeated detection of p-4EBP1 both in the cytoplasm and in the nucleus by us and other groups is an interesting finding (Zhou et al. 2004; Castellvi et al. 2006; Rojo et al. 2007). Contrary to us, other authors did not stringently separate nuclear and cytoplasmic staining for statistical analysis. However, Rojo et al. (2007) as well as Castellvi et al. (2006) noticed that cytoplasmic p-4EBP1 expression occurred in invasive breast and ovarian carcinomas as opposed to nuclear expression in normal epithelium and stromal cells. While the mechanistic background of 4EBP1 nuclear localization has not yet been described, it has been demonstrated recently that its target protein eIF4E has functions as a nuclear regulator of the export of several RNAs involved in proliferation and cell growth (Culjkovic et al. 2007). The immunohistochemical studies cited above point to a nuclear role of 4EBP1 and provide a rationale for further functional studies.

Supporting our finding of reduced levels of several elements of the mTOR pathway, such as p-mTOR, p-4EBP1, total 4EBP1 and AKT after mTOR inhibition, (Bae-Jump et al. (2006) observed a reduction in the protein levels of the mTORC1 downstream target total and activated S6 kinase-1 (S6K1) after rapamycin treatment of ovarian and cervical cancer cells. This suggests an effect of rapamycin on gene expression or degradation of mTOR up- and downstream effectors. Zhou et al. (2003) reported a reduction in the levels of phosphorylated 4EBP1 and S6 protein, while the levels of the unphosphorylated proteins remained stable. An explanation for this loss of (activated) effector proteins of the mTOR pathway under rapamycin therapy is lacking to date, but one might speculate that this effect might add to the efficacy of the inhibitor.

In our study, we did not observe a compensatory activation of AKT, in PTEN-deficient or in PTEN wild-type cells. In several other cell lines, increased AKT activation was detected after rapamycin treatment (Sabatini 2006). A current explanation is that mTORC1 in a negative feedback loop via IRS1 (insulin receptor substrate 1) inhibits AKT phosphorylation; mTORC1 inhibition results in increased AKT phosphorylation, which might compensate for the anti-proliferative function of rapamycin. This compensatory AKT activation seems to be cell type-specific and is held responsible for the varying success rate of mTOR inhibition in clinical trials (Sabatini 2006). Endometrial adenocarcinoma cells might belong to the category of cells in which this negative feedback loop is not relevant, which encourages the experimental use of mTOR inhibitors in this malignoma.

Several groups have found a growth inhibition and cell cycle arrest in G1 phase following mTOR inhibition of various cancer cells (Zhou et al. 2003; Gao et al. 2004), yet only few studies have reported endometrial cancer cell culture experiments with treatment using mTOR inhibitors. Zhou et al. (2003) reported a growth inhibition in both PTEN-deficient Ishikawa and HEC-1B and PTEN wild-type ECC endometrial cancer cells with rapamycin treatment for up to 6 days (Gao et al. 2004). Similarly, Treeck et al. (2006) treated PTEN wild-type HEC-1A cancer cells with the synthetic mTOR inhibitor RAD001 and observed an up to 50% growth inhibition with rather high doses of the inhibitor. In the light of these studies, our finding of a clear difference between PTEN status/AKT activation and response to rapamycin points to a varying influence of the AKT pathway dependent on the cell type. In our study, we further found that certain low-level mTOR activation could also be observed in PTEN wild-type cells even in the absence of activated AKT. In these cells, other pathways like the ras/raf/ERK pathway or hypoxia-induced HIF1α activation might induce mTOR activation (Sabatini 2006). This low-level mTOR activation might explain the weak but detectable effect of rapamycin on cell proliferation. For the situation in vivo one might speculate that PTEN loss might not by itself be predictive for the response of a certain endometrial adenocarcinoma to mTOR inhibition, but it might be more significant in connection with other markers. Potential new and more “direct” markers for therapy response might be the expression status of the mTOR cascade molecules.

In the current study, we present two potentially valuable markers for the determination of an in vivo mTOR activation in endometrial adenocarcinoma, p-mTOR and p-4EBP1. We have demonstrated that the investigation of these markers is feasible in human paraffin-embedded tumor tissue and provides interesting information on a link between tumor progression and mTOR pathway activity. A next step of the validation of the predictive effect of p-mTOR and p-4EBP1 expression would be the investigation of a larger number of cell lines in vitro on the one hand. On the other hand, it would be interesting to test our hypothesis in large-scale, and preferentially multicenter clinical studies comprising more high-risk endometrial cancers in vivo. It would further be interesting to examine the predictive function of these markers in tumor tissue from patients participating in a study evaluating the effect of mTOR inhibitors. In studies where sequential tissue sampling is possible, it would also be desirable to evaluate a potential AKT activation by mTOR inhibition directly in the tissue of treated patients.

As a conclusion, our results show that mTOR activation predominantly occurs in high-grade, high-stage endometrial adenocarcinomas as well as in endometrial cancer cells that respond well to a treatment with rapamycin. This points to the important role of the mTOR pathway in endometrial cancer progression. Our data provide a rationale for the further evaluation of the immunohistochemical detection of activated mTOR pathway components as a diagnostic tool for the selection of patients with an activated mTOR pathway, who could be considered for a therapy with mTOR inhibitors.

Acknowledgments

We would like to thank Mrs. Ines Koch and Mrs. Petra Wachs for their excellent technical assistance.

Footnotes

The authors Silvia Darb-Esfahani and Areeg Faggad contributed equally to the publication.

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