Introduction

Endometrial cancer is the most common cancer of the female reproductive tract with 150,000 new cases diagnosed annually worldwide [1]. Approximately, 90% of endometrial cancer cases are sporadic, whereas the remaining 10% are hereditary [2]. Pathogenesis studies of cancer on cellular functions including cell proliferation and survival revealed that the epidermal growth factor receptor (EGFR) plays a key role [3]. Cell growth depends on persistent activation of the EGFR pathway in the majority of human epithelial cancers [4], among which EGFR amplification has been described in around 60–80% of endometrial carcinomas [5, 6]. Moreover, poor prognosis indicators of high-grade endometrial carcinomas as deep myometrial invasion and poor survival rate of patients were observed when EGFR over expressed [7, 8]. Therefore, EGFR-targeted therapeutic agents are valuable and currently being investigated. However, EGFR tyrosine kinase inhibitor (TKI) resistance has been reported in several cancers. And further studies implied that the resistance was associated with the absence of PTEN (phosphatase and tensin homologue deleted on chromosome 10) [9, 10].

It has been well established that PTEN is the most frequently altered gene in the pathogenesis of type I endometrioid endometrial carcinoma [11]. The loss of PTEN function is an early event in endometrial tumorigenesis [12]. Mechanistically, PTEN is a PI3K antagonist that removes the 3′ phosphate of PIP3 and attenuates the activation of PI3K, hence inhibiting PI3K’s downstream targets mainly as protein kinase B (PKB/AKT), although other genes of cell survival and proliferation were also effected. In accordance with this, studies showed that PTEN reconstitution in PTEN-mutated endometrial carcinoma cells restored contact inhibition of cells that is accompanied by decreased phospho-AKT expression [13]. Others also found that mammalian target of rapamycin (mTOR) protein is an important downstream signaling mediator in PTEN-negative tumors [14, 15]. Thus, PTEN/PI3K/AKT/mTOR axis may contribute to the pathogenesis of endometrial carcinoma Besides intervening signaling pathway, PTEN also modulates cell cycling through increasing apoptosis and the G1 phase and decreasing the S and G2–M phases, which further sensitizing the cells to gefitinib [16].

Thus, the mechanism of EGFR inhibitor resistance was accordingly turned to PTEN’s downstream targets at the level of PI3K/AKT/mTOR regulation. On one hand, down-regulation of PI3K/AKT and MAPK signaling pathways were observed after anti-EGFR agents treatment [17]. On the other hand, PTEN loss leads to uncoupling increase of the PI3K/AKT signaling pathway from EGFR and results in EGFR kinase inhibitor resistance [9]. Hence targeting PI3K/AKT signaling pathway is the pivotal point to enhance the sensitivity of PTEN-deficient cancer cells to EGFR kinase inhibitors [9, 18]. As such, we hypothesized that blocking PI3K/AKT pathway by applying mTOR inhibitors could improve the sensitivity of endometrial cancer cells to EGFR inhibitor, and in consequence produce an antitumor effect.

To test this hypothesis, both a none PTEN expressed and a high PTEN expressed endometrial cancer cell lines are used. PTEN gene knockdown as well as reconstitution was performed to investigate the relationship between the PTEN expression level and TKI resistance. Our data demonstrated that mTOR kinase inhibitor could correct EGFR kinase inhibitor resistance in PTEN-deficient tumor cells.

Materials and methods

Cell culture

HEC-1A and Ishikawa human endometrial carcinoma cells were used in this study. Ishikawa cells do not express active PTEN protein due to missense mutations, whereas HEC-1A cells exhibit a high wild-type PTEN expression level. The two cell lines were maintained in Dulbecco’s modified Eagle’s medium (DMEM) that was supplemented with 10% fetal bovine serum under an atmosphere of 95% air and 5% CO2.

Reagents

RG14620, an inhibitor of EGFR tyrosine kinase, was provided by ENZO Life Sciences, Inc. (Plymouth Meeting, PA, USA). Rapamycin, which is an mTOR kinase inhibitor, rabbit anti-phosphorylated-EGFR (Tyr1068), anti-EGFR, anti-phosphorylated-AKT (Ser473), anti-AKT, anti-phosphorylated-mTOR, and anti-mTOR antibodies were all purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA). Anti-phosphorylated-p70S6, anti-p70S6, and anti-PTEN antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Production of lentiviral vectors and transduction into endometrial cancer cells

Two lentiviral vectors were constructed by GeneChem (Shanghai, China) as follows. One lentiviral-RNAi vector contained a RNA interference sequence against PTEN (PTEN-RNAi-LV), and the other lentiviral gene expression vector contained PTEN cDNA. The PTEN-RNAi-LV targeted the sequence “GTATAGAGCGTGCAGATAA” of the PTEN gene. The green fluorescent protein (GFP) expression cassette was inserted into the same vector as a reporter gene. The lentiviral vector for PTEN overexpression (PTEN-OV-LV) contains the entire ORF of PTEN (NM_000314) driven by the promoter of UbiC. The lentiviral particles were produced according to the instructions from Invitrogen. Briefly, the vectors with helper plasmids were transfected into 293T cells using the calcium phosphate transfection method. The supernatant containing lentiviral particles were collected and concentrated by ultracentrifugation. The condensed lentiviral particle solution was tittered on 293T cells with the final titer about 1 × 109 TU/ml. HEC-1A (5 × 104 cells/well) and Ishikawa cells (2 × 105 cells/well) were plated in 6-well plates overnight. HEC-1A cells were transfected with PTEN-RNAi-LV at a multiplicity of infection (MOI) of 50, and Ishikawa cells were transfected with PTEN-OV-LV at a MOI of 10. The expression of GFP or the PTEN genes was detected using a fluorescent microscope and western blotting 120 h after the cells were transfected.

Inhibitor treatments

The tyrosine kinase inhibitor RG14620 and the mTOR inhibitor rapamycin were used. These inhibitors were dissolved in DMSO and diluted using the culture medium. The culture medium containing the equivalent concentrations of DMSO served as vehicle controls.

Western blot analysis

HEC-1A, HEC-1APTENkd, Ishikawa, and IshikawaPTEN cells were plated at a density of 2 × 105 cells/6-cm dish 1 day prior to RG14620 and rapamycin treatment. Four cell lines were seeded and subjected to the following treatments: DMSO (vehicle, control), RG14620 (10 μM in DMSO), rapamycin (10 nM in DMSO), RG14620 (10 μM in DMSO), and rapamycin (10 nM in DMSO). Cells were lysed in a buffer containing 50 mM Tris (pH 7.4), 150 mm NaCl, 0.5% NP-40, 50 mm NaF, 1 mm Na3VO4, 1 mm phenylmethylsulfonyl fluoride, 25 mg/ml leupeptin, and 25 mg/ml aprotinin. The lysates were cleared by centrifugation, and the supernatants were collected. Equal amounts of protein lysates were used for western blot analysis with the indicated antibodies. Total proteins were measured using the Bradford method, and 20–25 μg of total protein was electrophoretically separated in 8–10% acrylamide gels (SDS–PAGE by Laemmli). Gene expression was detected using a chemiluminescence assay (ECL-Plus, Amersham Life Science, UK). The protein level was quantified using densitometric analysis with Quantity One software (BioRad, Hercules, CA), and β-actin expression levels were used to further normalize the loading amount.

Growth assay

Cell viability was determined by MTT assay (Sigma, USA). HEC-1A, HEC-1APTENkd, Ishikawa, and IshikawaPTEN cells were incubated in 96-well plates at a density of 5 × 103 cells/well. Twenty-four hours later, cells were treated with various concentrations of RG14620 in complete medium ranging from 0 to 100 μmol/l. A dose response curve following RG14620 treatment (0–100 μmol/l) was also conducted with 5 or 10 nmol/l rapamycin to study the effects of combined treatment. Forty eight hours after treatment cells were exposed for 4 h to sterile MTT dye (5 mg/ml). The supernatant was discarded, and a 150 μl of dimethyl sulfoxide was added and mixed thoroughly for 10 min. The spectrometric absorbance at a wavelength of 490 nm was measured using an enzymatic immunoassay analyzer (model 680; Bio-Rad, USA). Triplicate wells for each dose were conducted and the experiments were performed three times independently.

Cell cycle and apoptosis analyses

HEC-1A, HEC-1APTENkd, Ishikawa, and IshikawaPTEN cells were seeded and subjected to four different treatments with DMSO (vehicle, control), RG14620 (10 μM in DMSO), rapamycin (10nM in DMSO), RG14620 (10 μM in DMSO), and rapamycin (10nM in DMSO). After being treated for 48 h, the cells were harvested by trypsinization and centrifuged at a speed of 400×g for 5 min. A kit from BD was used for the cell cycle analysis. The supernatant was carefully decanted and 250 μl of solution A (trypsin buffer) was added for digestion for 10 min. A total of 200 μl of solution B (trypsin inhibitor and RNase buffer) was added for neutralization for 10 min. Samples were incubated with 200 μl of cold solution C (propidium iodide stain solution) in the darkness at 4°C for 10 min. The cell cycle was analyzed using flow cytometry and FACSDiva software. For the detection of apoptosis, cells were stained with Annexin V-PE and 7-amino-actinomycin (7-AAD). Cells were washed with cold PBS twice and then re-suspended in 1× binding buffer at a concentration of 105–106 cells/ml. A total of 100 μl of cell suspension (1 × 105 cells) was transferred to a 5 ml culture tube. In total, 5 μl of Annexin V-PE and 5 μl of 7-AAD were added to the cell suspension and incubated for 15 min in the darkness at room temperature. Another 400 μl of the binding buffer was added before the samples were analyzed by using a FACScan flow cytometer with the FACSCalibur system. All of the experiments were performed in triplicates.

Statistical methods

The data were presented as the mean ± SD. Student’s two-tailed t test was used to compare the values of the test and control samples. A value of P < 0.05 was considered statistically significant.

Results

Modulating PTEN expression in endometrial cancer cells regulated the expression and activation of EGFR and AKT/mTOR signaling

We stably transfected HEC-1A and Ishikawa cells with various lentiviral vectors to create several cell lines with various expression levels of the PTEN protein. In addition, GFP was incorporated as a reporter gene. After a single exposure of HEC-1A and Ishikawa cells to the encoding lentivirus, more than 95% of the transfected cells expressed GFP at 120 h after the transfection, indicating high transfection efficiency. As shown in Fig. 1a and b, a significant silencing effect was obtained from PTEN-RNAi-LV, whereas the empty plasmid had no effects. The transfection of wild-type PTEN into Ishikawa cells dramatically increased PTEN expression, whereas an empty plasmid had no effect (Fig. 1c, d).

Fig. 1
figure 1

Western blot analysis of PTEN expression in HEC-1A and Ishikawa cells after stable transfection with PTEN-RNAi or wild-type PTEN, respectively. The bar graph shows the PTEN expression of endometrial cancer cells after stable transfection (compared with untreated and control vector groups; *P < 0.01). The results are representative of three independent experiments

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To examine the interaction between the PTEN status and the activation of EGFR and AKT/mTOR signaling in vitro, we performed western blot analysis to examine the expression and phosphorylation of EGFR, AKT, and mTOR in endometrial cancer cells. As shown in Fig. 2, silencing PTEN in HEC-1A cells increased the expression and phosphorylation of EGFR and AKT compared with parental cells and promoted downstream mTOR phosphorylation, which enhanced cell proliferation. PTEN re-expression in Ishikawa diminished the expression of EGFR and AKT and the phosphorylation of EGFR, AKT, and mTOR compared with parental cells, which suppressed cell growth.

Fig. 2
figure 2

Western blot analysis of EGFR, phospho-EGFR, mTOR, phospho-mTOR, AKT, and phospho-AKT expression in HEC-1A and Ishikawa cells after transfection with PTEN-RNAi or wild-type PTEN (a). The bar graph shows protein expression profiles of untransfected and transfected (b) HEC-1A cells and (c) Ishikawa cells (compared with untreated and control vector group: *P < 0.01). The results are representative of three respective experiments

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Cell viability was influenced by PTEN expression levels in vitro

To further address if the crosstalk between PTEN and EGFR has biological function significance, we examined PTEN effects on cell proliferation. As shown in Fig. 3a, the viability of HEC-1A cells was increased compared to untreated and control groups at 72 h after PTEN-RNAi-LV transfection. However, the viability of Ishikawa cells decreased at 48 and 72 h after PTEN-OV-LV transfection (Fig. 3). Therefore, PTEN expression could inhibit cell proliferation accompanied by EGFR protein expression reduction.

Fig. 3
figure 3

Cell viability was measured using MTT assays at 24, 48, and 72 h after PTEN-RNAi-LV and PTEN-OV-LV transfection in HEC-1A and Ishikawa cells. The results shown are one representative experiment from three experiments with similar results. Each point represents the mean ± SD of three independent experiments. The significance was determined using the Student’s t test (*P < 0.05, **P < 0.01 compared with untreated control)

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EGFR inhibitor sensitivity was dependent on PTEN status in vitro and was enhanced by rapamycin treatment in endometrial cancer cells, especially in PTEN-deficient cells

HEC-1A, HEC-1APTENkd, Ishikawa, and IshikawaPTEN cells were treated with 10 μmol/l RG14620, 10 nmol/l rapamycin, or a combination of both inhibitors in serum-containing complete medium for 2 days. RG14620 monotherapy diminished the phosphorylation of EGFR in four mock-transfected and transfected cell lines (P < 0.05). However, the phosphorylation of AKT and p70S6 was reduced in HEC-1A and IshikawaPTEN cells, which expressed PTEN protein (P < 0.05). Rapamycin exposure decreased phosphorylated p70S6 kinase levels in the mock-transfected and transfected cell lines (P < 0.05). The presence of rapamycin in combination with RG14620 significantly inhibited the phosphorylation of AKT and p70S6, particularly in cells lacking PTEN (P < 0.05) (Fig. 4).

Fig. 4
figure 4

Western blot analysis showing the phosphorylation protein levels in endometrial carcinoma cells with different PTEN statuses that were exposed to RG14620, rapamycin, or a combination of both inhibitors for 48 h. RG14620 decreased phospho-EGFR expression in mock-transfected or transfected cells. RG14620 decreased phospho-AKT and phospho-p70S6 expression in PTEN-positive cells such as HEC-1A and IshikawaPTEN cells and showed no effect on PTEN-negative Ishikawa and HEC-1APTENkd cells. Rapamycin decreased phospho-p70S6 expression in these four cell lines, and the combination of RG14620 with rapamycin significantly decreased phospho-AKT and phospho-p70S6 expression in PTEN-negative cells. (*P < 0.05, compared with control. # P < 0.05, compared with RG14620 or rapamycin-treated group.) The results are representative of three independent experiments

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The dose response of endometrial cancer cell lines to RG14620 or a combination of RG14620 and rapamycin was determined using MTT assays. HEC-1A, HEC-1APTENkd, Ishikawa, and IshikawaPTEN cells were continuously exposed for 48 h to increasing concentrations of RG14620 and 0, 5, and 10 nmol/l rapamycin. HEC-1A cells did display sensitivity to RG14620 with an IC50 value of 30.8 ± 3.6 μM. After silencing PTEN expression, the HEC-1APTENkd cells were resistant to RG14620 with an IC50 value of 162.5 ± 21.4 μM. In contrast, PTEN reintroduction sensitized IshikawaPTEN cells to RG14620 (IC50 value of 38.0 ± 9.3 μM compared with 170.3 ± 14.4 μM in Ishikawa cells) (Table 1).

Table 1 IC50 values of RG14620 with various concentrations of rapamycin for 48 h in HEC-1A, HEC-1APTENkd, Ishikawa, and IshikawaPTEN cells. IC50 was determined using MTT assay
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When combined with 5 nmol/l of rapamycin, the IC50 value of RG14620 was greatly reduced in HEC-1APTENkd cells (32.8 ± 6.2 μM) and Ishikawa cells (33.7 ± 5.4 μM). Concurrent administration of 10 nmol/l rapamycin with various RG14620 doses showed a synergistic effect and lowered the RG14620 IC50 values in all cell lines (with IC50 values of 21.4 ± 3.4 μM in HEC-1A cells, 14.1 ± 1.6 μM in HEC-1APTENkd cells, 14.0 ± 3.2 μM in Ishikawa cells, and 20.0 ± 1.6 μM in IshikawaPTEN cells), especially in PTEN-deficient cell lines. This result is not only attributable to additive growth inhibitory effects of the two drugs. The addition of 5 nM rapamycin alone has no significant growth inhibitory effects. However, it sensitizes Ishikawa and HEC-1APTENkd cell lines to RG14620. This finding indicated that rapamycin sensitized PTEN-deficient and PTEN-intact endometrial cancer cells to RG14620, particularly in PTEN-deficient cells (Fig. 5; Table 1).

Fig. 5
figure 5

Dose responses of endometrial cancer cell lines that were exposed to different concentrations of RG14620 combined with or without various concentrations of rapamycin for 48 h were determined using MTT assay. The down-regulation of PTEN expression sensitized the HEC-1A cells to RG14620, which was indicated by the decrease in the IC50 value from 162.5 ± 21.4 to 30.8 ± 3.6 μM. PTEN reintroduction in Ishikawa cells sensitized the cells to RG14620, which was indicated by the decrease in the IC50 value from 170.3 ± 14.4 to 38.0 ± 9.3 μM. PTEN deficiency in cells such as HEC-1APTENkd and Ishikawa cells facilitated a synergistic effect between rapamycin and RG14620, which was demonstrated by the decreased IC50 value

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Inhibition of EGFR and mTOR signaling pathways significantly enhanced apoptosis in PTEN-deficient and PTEN-intact cells

To further understand how rapamycin prevents EGFR inhibitor resistance, we evaluated the percentage of apoptotic cells identified by Annexin V-PE/7-AAD staining 48 h after same treatment as shown above. RG14620 alone promoted significant apoptosis in PTEN-intact HEC-1A and IshikawaPTEN cells (P < 0.01), but not in PTEN-deficient HEC-1APTENkd and Ishikawa cells compared to DMSO controls (P > 0.05). In contrast, rapamycin enhanced apoptosis in PTEN-deficient HEC-1APTENkd and Ishikawa cells (P < 0.01) but not in PTEN-intact HEC-1A and IshikawaPTEN cells (P > 0.05). Irrespective of these, both PTEN-deficient and PTEN-expressing cells exhibited significantly increased apoptosis after being treated with the two inhibitors compared with either of the monotherapy (P < 0.05) (Table 2; Fig. 6).

Table 2 Effects of RG14620 and rapamycin alone or in combination on apoptosis of HEC-1A, HEC-1APTENkd, Ishikawa, and IshikawaPTEN cells. Apoptosis was measured as percentage of apoptosis cells
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Fig. 6
figure 6

Comparison of apoptosis between untreated and treated cells. a Percentage of apoptotic cells (stained with Annexin V-PE and 7-AAD and analyzed by FACS) after treatment with 10 μM RG14620, 10 nM rapamycin, or a combination of both inhibitors. b Cumulative histograms of apoptotic induction. (*P < 0.05, compared with untreated control; # P < 0.05, compared with RG14620 or rapamycin-treated group.) The results are representative of three separate experiments

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Single versus combined treatment with RG14620 and rapamycin altered the cell cycle in endometrial cancer cells with different PTEN expression levels

Then we continuously to analyze another aspect of the antiproliferative activities of RG14620 and rapamycin in endometrial cancer cells: cell cycle. HEC-1A and IshikawaPTEN cells but not HEC-1APTENkd and Ishikawa cells treated with 10 μM RG14620 for 48 h displayed a significant increase in the G1 phase and a reduction in the S phase compared to the control. Treatment with 10 nM rapamycin resulted in a significant increase in the G1 phase and a decrease in the S phase in HEC-1APTENkd and Ishikawa cells but not HEC-1A and IshikawaPTEN cells. The combined treatment with RG14620 and rapamycin in four transfected and mock-transfected cell lines increased the G1 phase and decreased the S phase compared with monotherapy with RG14620 or rapamycin. Taken together, these results show that RG14620 and rapamycin cause cell cycle alterations with G1 arrest (Fig. 7).

Fig. 7
figure 7

Effect of RG14620 and/or rapamycin on the cell cycle distribution of endometrial cancer cells. HEC-1A, HEC-1APTENkd, Ishikawa, and IshikawaPTEN cells were treated with 10 μM RG14620, 10 nM rapamycin, or a combination of both inhibitors, fixed, permeabilized, stained with PI, and analyzed by flow cytometry. The bar diagrams indicate the distribution of the cells in the different phases of the cell cycle

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Discussion

EGFR, which is a member of the HER family of receptor tyrosine kinases, has been shown to be highly expressed in endometrial cancers. Much attention has been focused on EGFR-targeted therapies such as tyrosine kinase inhibitors [19]. Previous studies have indicated that tumor cells with mutated PTEN are insensitive to selective inhibitors of EGFR. The dysfunction of PTEN leads into a less-sensitive phenotype to peptide growth factors via constitutive activation of the PI3K/AKT/PKB signaling pathway in endometrial carcinoma [20]. But additional mechanisms contributing to EGFR inhibitor resistance in endometrial cancer remain elusive. Our previous studies indicated that PTEN expression affected the sensitivity of endometrial carcinoma cells to relate signal transduction inhibitors [21, 22]. In the current study, we demonstrated that there is crosstalk between EGFR signaling and the function of the tumor suppressor PTEN in endometrial cancer. Our results support a notion that the expression status of PTEN is likely to affect the effectiveness of therapies using an EGFR inhibitor alone or a combination of EGFR and mTOR inhibitors.

In several study, re-established PTEN expression in Ishikawa cell lines or knocked down PTEN in HEC-1A cells was used to study the role of PTEN with cell signaling and drug resistant [23–25]. To study the role of PTEN with EGFR and AKT/mTOR signaling, we used HEC-1A and Ishikawa cells that had different PTEN functionalities as a model. We stably silenced PTEN expression in HEC-1A cells using a lentivirus containing short hairpin RNAs against PTEN and reintroduced the PTEN gene in Ishikawa cells using lentiviral transfection. The PTEN transgene suppressed cell growth through induction of apoptosis in cells lacking wild-type PTEN [23, 26]. Consistent with the previous studies [23], PTEN negatively regulates AKT, phospho-AKT, and phospho-mTOR in our system in both gain and loss function studies. More than this, we found that the PTEN knockdown also increase the expression of EGFR, phospho-EGFR in HEC-1A cells, where as PTEN reconstitution decreased their expression in Ishikawa cells. It suggested a crosstalk between PTEN and EGFR signaling pathway. PTEN upregulation significantly inactivated EGFR but not the AKT/mTOR pathway. In contrast, when the PTEN gene was knocked down, the PI3K/AKT/mTOR pathway and EGFR were activated.

This crosstalk happens at the level of PI3K/AKT/mTOR pathway, which may indicate the molecular mechanisms of EGFR TKI resistance, which associated with a high level expression of AKT/mTOR. This serves as a theoretical foundation for the combination therapy. Our study indicated that PTEN status affected the sensitivity of endometrial cancer to EGFR TKIs. We treated four transfected and mock-transfected cell lines with RG14620, which is an EGFR TKI. We demonstrated that the RG14620 IC50s values in PTEN-intact cells were significantly lower than those in PTEN-deficient cells. RG14620 effectively induced G1 arrest and apoptosis in PTEN-intact cells compared with PTEN-deficient cells. Although RG14620 effectively inhibited EGFR phosphorylation in PTEN-deficient cells, these cells remained relatively resistant to RG14620 and did not display AKT/mTOR pathway inhibition. However, RG14620 inhibited EGFR phosphorylation and AKT/mTOR pathway activation in PTEN-intact cells. We showed that the resistance of endometrial cancer cells to EGFR TKIs was caused by PTEN deficiency with consequent hyperactivation of EGFR and AKT/mTOR, which is partially mediated by uncoupling of the AKT/mTOR pathway from EGFR. Reconstitution of PTEN function reestablished EGFR-stimulated AKT/mTOR signaling and restored EGFR TKI sensitivity in PTEN-intact cells.

One of the main functions of PTEN is to counteract the activity of the PI3K/AKT/mTOR pathway. Therefore, pharmacologic inhibitors targeting the PI3K/AKT/mTOR signaling pathway downstream of PTEN may mimic the effects that are rescued by PTEN restoration. The protein mTOR is an important downstream signaling mediator in PTEN-negative tumors [27, 28]. PTEN status affected the sensitivity of endometrial cancer to mTOR inhibitors. PTEN-intact tumor was resistant to rapamycin conferring to PTEN-deficient tumor. Suppression of PTEN function increased endometrial cancer mTOR inhibitor sensitivity thought activating AKT/mTOR signal pathway, as previously reported [29]. Our study indicated that rapamycin enhanced PTEN-deficient cell sensitivity to RG14620. The combination of EGFR/mTOR inhibitors promotes cell death, G1 arrest and apoptosis synergistically in PTEN-deficient cells. We also detected that RG14620 and rapamycin co-treatment had additive effects on the inhibition of the downstream AKT/mTOR pathway by dramatically inhibiting the phosphorylation of AKT and p70S6 in cells lacking PTEN. These results demonstrate that treatment of PTEN-deficient cells with an mTOR inhibitor similarly restores EGFR-stimulated AKT/mTOR signaling and re-sensitizes these cells to EGFR TKI.

It is known that the combined therapy with EGFR TKI and the mTOR inhibitor provides synergistic benefits by promoting cell death in PTEN-deficient tumor cells and PTEN-intact tumor cells [18]. In our experimental model, the combination therapy also promoted an additive effect on G1 arrest and apoptosis over the single therapies. In PTEN-intact tumor cells, the addition of rapamycin might allow for a complete inhibition of mTOR signaling. We demonstrated that the additive effect of combination therapy were more dramatic in PTEN-deficient cells with elevated EGFR activation. Because EGFR was activated after PTEN gene silencing, EGFR TKI may be more effective in PTEN-deficient than PTEN-intact endometrial cancer cells when combined with the mTOR kinase inhibitor.

In summary, we showed that PTEN deficiency promoted EGFR TKI resistance, in part by dissociating EGFR inhibition from downstream PI3K/AKT/mTOR pathway inhibition. If the mTOR kinase signaling is inhibited, cells are more dependent on the EGFR pathway for survival. Inhibition of EGFR and mTOR kinase signaling by combinatorial drug therapy may be useful for the treatment of PTEN-deficient tumor cells. These results have significant predictive and therapeutic clinical implications and identify PTEN status as a marker of EGFR inhibitor sensitivity. Patients with PTEN-intact tumors may be sensitive to EGFR TKIs, whereas patients with PTEN-null tumors are unlikely to respond to this treatment, which can be reversed by PTEN reintroduction. In addition, our results also suggest that the co-treatment of EGFR inhibitors with mTOR kinase inhibitors in patients with PTEN-null tumors may decrease EGFR TKI resistance and provide greater benefits in these patients compared to the treatment of patients with PTEN-intact tumors. Therefore, a priori screening of endometrial tumors for PTEN expression status may help identify patients who are likely to benefit from these therapies. Validation of these hypotheses requires additional clinical correlative studies.