Reciprocal Negative Regulation between EGFR and DEPTOR Plays an Important Role in the Progression of Lung Adenocarcinoma

As one of the most common cell-surface receptors that specifically bind with extracellular protein ligands of the epidermal growth factor (EGF) family, the epidermal growth factor receptor (EGFR) belongs to the ErbB family of receptors and is involved in different types of diseases, including age-related diseases, autism spectrum disorders, and cancers (1–3). In light of the confirmed evidence that EGFR plays a pivotal role in tumorigenesis, drug resistance, relapse, and the metastasis of various cancers (4–7), some of EGFR tyrosine kinase inhibitors (TKIs) or monoclonal antibodies have been developed and entered clinical trials as cancer therapeutics, such as afatinib, gefitinib, and erlotinib (www.fda.gov; refs. 8, 9).

In lung adenocarcinoma, EGFR is overexpressed in more than 39% and has emerged as a leading target for the treatment of patients with adenocarcinoma (10–12). Some TKIs, like gefitinib, significantly improved survival and prognosis of patients with lung adenocarcinoma, while some patients acquired resistance after initial response to TKIs or even had intrinsic resistance without EGFR mutations (13). To date, the possible mechanisms that underlie the primary resistance of these drugs have been supposed as follows: (i) a result of somatic mutations occurring within the tyrosine kinase domain of EGFR (14, 15), the phosphorylation of EGFR triggered several downstream signal transduction cascades, such as the MAPK, Akt/mTOR, and JNK pathways, which almost totally inhibited by gefitinib, while mutated receptors sustain a hyperactivated downstream signaling (16–18); (ii) activation of other signaling pathways alternative to EGFR, such as the insulin-like growth factor 1 receptor (IGF-1R) and vascular endothelial growth factor receptor (VEGFR; ref. 19), and resulted in the upregulation of downstream signaling pathways such as AKT/mTOR (20–23). Therefore, it is of great significance to further explore the downstream pathways of EGFR and thus develop novel therapeutic strategies for patients with lung adenocarcinoma.

The mammalian target of rapamycin (mTOR) is a highly conversed serine-threonine kinase that is located in the PI3K/AKT pathway and is frequently hyperactivated in tumors by mutations in upstream signaling factors (e.g., EGFR, VEGFR, and PI3K regulators; refs. 23, 24). Various mTOR inhibitors have been used in trials for cancer therapeutic (25, 26). DEP domain-containing mTOR-interacting protein (DEPTOR), as a naturally occurring inhibitor of mTOR, usually acts as a tumor suppressor by blocking the activation of mTOR to inhibit cell proliferation, invasion, and survival effect of AKT (27, 28). However, under certain circumstances, DEPTOR could act as an oncogene by relieving the feedback inhibition of mTOR, activation of mTOR enhances the downstream S6K expression, which could negatively regulate AKT via downregulating expression of IRS-1/2 (27, 29). More importantly, mTOR could also cooperate with S6K or CK1α to enhance βTrCP to degrade the expression of DEPTOR and thus generate an autoamplification loop to promote its own full activation (27, 30).

Although the potential role of DEPTOR as an oncogene or a tumor suppressor has been investigated in different types of tumors (28, 31–33), and DEPTOR could inhibit proliferation of lung adenocarcinoma cells (34), it has not been previously tested whether and how DEPTOR plays a role in EGFR-induced tumor progression of lung adenocarcinoma. In this study, we showed that DEPTOR expression is significantly reduced in patients with lung adenocarcinoma and negatively correlated with EGFR expression. Activation of EGFR signaling with EGF in EGFR overexpressing A549 cells dramatically inhibited DEPTOR expression by enhancing the function of the mTOR autoamplification loop, which mainly responds to the feedback degradation of DEPTOR. Furthermore, DEPTOR knockout by using the CRISPR/Cas9 system significantly promoted tumor growth both in vivo and in vitro. And, more importantly, tumor progression induced by EGFR ectopic expression was significantly inhibited by simultaneously transfected with DEPTOR-overexpressing plasmid. Taken together, our results reveal a novel mechanism that EGFR used to facilitate tumor growth and DEPTOR may be a novel therapeutic option for the treatment of human lung adenocarcinoma.

The study was approved by the Ethical Review Board for research in the Zhongnan Hospital of Wuhan University. Fifty-one patients diagnosed as lung adenocarcinoma were given informed consent according to institutional guidelines. Biopsy specimens and paired adjacent normal tissues of all patients were acquired and immediately stored at liquid nitrogen until use. The patients' characteristics are summarized in Table 1. 

The normal human lung epithelial cell line BEAS-2B and lung adenocarcinoma cell line A549, PC-9 and NCI-H1975 were obtained from Shanghai Bogoo Biotechnology Limited, and cultured in RPMI-1640 medium (Gibco) supplemented with 10% fetal bovine serum (Gibco) at 37°C in a humidified atmosphere containing 5% CO₂. BEAS-2B cells were cultured in BEBM medium according to ATCC protocols.

Biopsy samples were fixed in 4% formalin and embedded in paraffin. Tissue slices were cut into 5-μm thick for H&E staining and examined under a microscope. Immunohistochemistry was performed using the Vectastain ABC Kit (Rabbit IgG, Vector Laboratories; ref. 34). Rabbit primary antibodies for DEPTOR (1:500), EGFR (1:200), phospho-EGFR (Tyr1068, 1:50) were purchased from Abcam. Slices were developed with DAB and counterstained with hematoxylin.

Total RNA of tumor tissues or cells was extracted using TRIzol (Invitrogen) according to the manufacturer's instructions. RNA was reversely transcribed into cDNA using the First-Strand cDNA Synthesis Kit (Toyobo). The mRNA expression of EGFR and DEPTOR was quantified using a real-time RT-PCR with the SYBR Green real-time PCR Master Mix kit (Toyobo). The following primers were used: EGFR, 5′-TGATAGACGCAGATAGTCGCC-3′ and 5′-TCAGGGC ACGGTAGAAGTTG-3′ (35); DEPTOR, 5′-TTTGTGGTGCGAGGAAGTAA-3′ and 5′-CATTG CTTTGTGTCATTCTGG-3′; GAPDH, 5′-CTCTCTGCTCCTCCTGTTCGAC-3′ and 5′-TGAGCGATGTGGCTCGGCT-3′ (31). The ABI StepOne Plus (Applied Biosystems) was used to perform the amplification reaction. Each experiment was performed in triplicate. And the date was analyzed by the 2^−ΔΔCt method.

Total proteins of tissue samples were extracted by using the Total Protein Extraction Kit (Millipore). As for cultured tumor cells, the cells were lysed with RIPA Lysis Buffer (Beyotime). Equivalent proteins (30 μg per sample) were electrophoresed in SDS-polyacrylamide gel and transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore). The PVDF membranes were blocked with 5% nonfat milk in TBST buffer for 1 hour and incubated overnight at 4°C with primary antibodies. The following rabbit antibodies were used: DEPTOR (Abcam), EGFR (Abcam), EGFR (Tyr1068; Abcam), mTOR, mTOR (Ser2448), CK1α, βTrCP1, S6K, S6K (Thr389), and β-actin (all from Cell Signaling Technology). Signals were detected using ECL detection reagent (Millipore) following the manufacturer's instructions.

Full-length EGFR and DEPTOR were cloned into the vehicle vector pcDNA3.1 to generate EGFR and DEPTOR overexpressing plasmids, as we previously described (1, 27, 31). A549 cells were transiently transfected with the above three plasmids using Lipofectamine 2000 according to the manufacturer's instructions (Invitrogen) and retained in a medium containing 10% FBS and 1 μg/mL puromycin to select the stable transfected cells.

A549 cells (2 × 10⁵) in 3 mL antibiotic-free medium were plated in 6-well plate. Twenty-four hours later, after cells reach 80% confluency, DEPTOR CRISPR/Cas9 KO Plasmid and DEPTOR HDR Plasmid (Cat. No. sc-402253 and sc-402253-HDR from Santa Cruz Biotechnology, Inc.) were mixed at equivalent ratio and then cotransfected into A549 cells using Lipofectamine 2000 according to the manufacturer's instructions. Forty-eight hours after transfection, aspirate the medium and replace with fresh medium containing puromycin (1 μg/mL) to select the stable transfected cells.

A cell proliferation assay was carried out with a Cell Counting Kit-8 (Beyotime; ref. 36). A549 or stable transfected cells were plated in 96-well plates at approximately 2 × 10³ cells per well. The numbers of cells per well were detected by the absorbance (450 nm) of reduced WST-8 at the indicated time points. The absorbance (450 nm) was measured by using SpectraMax i3x microplate reader (Molecular Devices).

Cell migration was determined by a wound-healing assay, and cells were seeded into 12-well plate until reached 90% confluence. Cell monolayers were carefully scratched using a sterile 200-μL pipette tip, and the cellular debris was subsequently removed by washing with PBS to form wound gaps (37). The wound location was marked, and cells were photographed at 0 and 24 hours to measure the cell migration ability by using an IX70 microscope (Olympus GmbH).

Cell invasion was determined by the Transwell assay. A total of 3 × 10⁴ cells in 0.5% FBS medium were transferred on the top of the Matrigel-coated invasion chambers (8-μm pore; Corning Costar), and 10% FBS medium was added in the lower chamber. After 48 hours of incubation, noninvasion cells were removed by wiping with a cotton swab. The invading cells that had invaded the bottom side of the membrane were then fixed with 4% formaldehyde and stained with 4′,6-dia-midino-2-phenylindole (DAPI; ref. 38). The numbers of invasive cells were obtained by counting five fields (×100) per membrane and represented the average of three independent experiments.

Six-to-eight-week-old female BALBc/nude mice were purchased from the Laboratory Animal Center of Wuhan University (Wuhan, China) and maintained in cages under sterile conditions with a specific pathogen-free environment. A549 cells, EGFR-overexpressing A549 cells, or EGFR/DEPTOR double overexpressing cells (1 × 10⁶) were injected subcutaneously within a volume of 100 μL in the flank at the time of inoculation (39). Tumor size was measured by external caliper measurement every three days after injection, and tumor volume was calculated as 1/2 × (tumor length) × (tumor width)² (40). Mice were sacrificed, and tumors were dissected and measured on day 21 after inoculation.

All data are presented as mean ± SD and analyzed by using Graphpad Prism V.5.00 software (GraphPad Software). A Spearman correlation test was used to assess the association between mRNA expression of EGFR and DEPTOR in tumor tissues. A comparison between two groups for statistical significance was performed with an unpaired Student t test. For more groups, one-way ANOVA followed by Neuman–Keuls post hoc test was used. P < 0.05 was considered statistically significant.

The abnormal hyperactivity of EGFR and its downstream mTOR pathways is the leading cause of resistance to TKIs; furthermore, mTOR inhibitors could effectively control the growth of lung adenocarcinoma even after acquiring resistance to TKIs (13, 41, 42). In order to further unveil the downstream regulatory molecule mechanisms involved in EGFR/mTOR activation, we simultaneously analyzed mRNA expression of EGFR and DEPTOR, a naturally occurring inhibitor of mTOR, in tumor tissues of patients with lung adenocarcinoma. As shown in Fig. 1A, the mRNA level of EGFR is significantly upregulated in lung carcinoma tissues than in adjacent normal compartment, and DEPTOR is downregulated in tumor tissues (Fig. 1B). In addition, this result is further confirmed by Western blotting (Fig. 1D) and immunohistochemistry analysis (Fig. 2A). Next, we then examined potential correlativity between aberrant EGFR and DEPTOR expression, Pearson correlation assay revealed that mRNA expression of EGFR significantly and negatively correlated with DEPTOR expression in tumor tissues (r² = 0.6247, P < 0.0001; Fig. 1C). As DEPTOR has been reported to inhibit tumor cell growth of lung adenocarcinoma (34), these findings suggest that there may be a link between EGFR and DEPTOR to regulate tumor growth.

Next, we detected EGFR and DEPTOR expression in three lung adenocarcinoma cell lines, including A549, PC-9, and NCI-H1975. Compared with normal human lung epithelial cells, BEAS-2B, protein (Fig. 2B) and mRNA (Fig. 2C) expression of EGFR were significantly increased in these tumor cells, and DEPOR expression was decreased at different levels (Fig. 2B and D). As A549 cells occupy the lowest level of DEPTOR expression and a relative higher level of EGFR expression, we then used A549 cells in our following experiments.

A549 cells were transfected with pcDNA3.1-EGFR to construct stable EGFR overexpressing cell line, and in comparison with empty vector, overexpression of EGFR significantly decreased DEPTOR expression in A549 cells (Fig. 3A). Previous studies revealed that EGF could induce the activation of EGFR kinase activity, and Gefitinib is selective EGFR TKI that usually used to block the EGFR signaling (43, 44). Herein we treated EGFR overexpressing A549 cells with these two reagents to test the effect of EGFR activation on DEPTOR expression. Our data showed that EGF (20 ng/mL) strongly stimulated the phosphorylation of EGFR and inhibited the expression of DEPTOR, while gefitinib (5 μmol/L) inhibited the phosphorylation of EGFR and significantly enhanced DEPTOR expression (Fig. 3B).

As studies have identified that S6K, CK1α, and βTrCP1 could be cooperated with mTOR to generate an autoamplification loop and thus trigger the degradation of DEPTOR (27, 30), we then detected the expression of these proteins with the presence of EGF or gefitinib. EGF significantly enhanced the phosphorylation of mTOR and S6K, as well as the expression of CK1α and βTrCP1 (Fig. 3C). Furthermore, blocking the activation of EGFR with gefitinib showed the opposite function on these four proteins' expression with EGF (Fig. 3D). Thus, we confirmed that EGFR activation enhances the function of mTOR autoamplification loop to downregulate DEPTOR expression.

Given that EGFR inhibited DEPTOR expression in A549 cells, we next investigated the potential roles of DEPTOR in lung adenocarcinoma development. First, we constructed two stable A549 cell lines, DEPTOR overexpressing cells and DEPTOR knockout cells, by using plasmid transfection or DEPTOR CRISPR/Cas9 system (Fig. 4A). Compared with empty vector control, overexpression of DEPTOR significantly decreased cell proliferation (Fig. 4B) and resulted in reduced abilities of migration (Fig. 4C and D) and invasion (Fig. 4D and F). However, in DEPTOR knockout cells, these tumor-related activities were all significantly enhanced as compared with those of parental control cells (Fig. 4B–F). Thus, these results confirmed that DEPTOR acts as a tumor suppressor in the tumorigenesis of lung adenocarcinoma.

Because EGFR has been found to widely overexpress and promote tumor progress in lung adenocarcinoma (15, 17), we consider whether DEPTOR ectopic expression could reverse the tumor- promoting effects of EGFR. EGFR overexpressing A549 cells were then further stably transfected with pcDNA-DEPTOR plasmid. As shown in Fig. 5A, pcDNA-DEPTOR plasmid transfection significantly elevated DEPTOR expression in EGFR overexpressing A549 cells. Simultaneously enforcing DEPTOR expression in EGFR ectopic expression A549 cells markedly decreased EGFR expression and its phosphorylation level. Analysis of tumor-related activities further revealed that DEPTOR inhibited the cell proliferation (Fig. 5B), migration (Fig. 5C and E), and invasion (Fig. 5D and F) of EGFR ectopic expression in A549 cells. Thus, these results demonstrated that ectopic expression of DEPTOR reverses EGFR-driven tumorigenesis in lung adenocarcinoma cells.

We noted that ectopic expression of DEPTOR significantly suppressed EGFR expression and its phosphorylation (Figs. 4A and 5A). To determine if protein synthesis was involved in DEPTOR-induced suppression of EGFR, cycloheximide (5 μg/mL) was used to inhibit protein synthesis, which at this dose has been shown to have an inhibitory effect on protein translation (45, 46). As shown in Fig. 6A and B, ectopic expression of DEPOR significantly promoted the degradation of EGFR protein. To further confirm the role of DEPTOR on mRNA expression of EGFR, the mRNA expression levels of EGFR in DEPTOR overexpression or knockout cells were detected, and there is no detectable difference in EGFR mRNA expression in response to DEPTOR expression (Fig. 6C). Next, actinomycin D (5 μg/mL; refs. 47, 48) was used to block mRNA transcription and then EGFR mRNA half-life was evaluated. Followed with actinomycin D treatment, no significant difference was seen in the rate of EGFR mRNA decay in response to DEPTOR expression (Fig. 6D). These results revealed that DEPTOR provides a feedback promotion on protein degradation of EGFR and does not influence EGFR mRNA transcription. So there is a reciprocal negative regulation between DEPTOR and EGFR expression.

The inhibitory effect of DEPTOR on the growth of lung adenocarcinoma cells in vivo was evaluated by using nude mouse models. As pcDNA empty vector stable transfection showed no detectable influence on EGFR and DEPTOR expression, as well as the proliferation, migration and invasion of A549 cells (Fig. 5), we used normal A549 cells as a control. EGFR overexpressing A549 cells and EGFR/DEPTOR double overexpressing A549 cells were injected into the flank of nude mice. The volume of tumor xenografts was monitored every 3 days. At day 21, mice were sacrificed, and the tumor xenograft of each mouse was dissected (Fig. 7A). As compared with normal control (214 ± 66 mm³), EGFR overexpressing A549 cells significantly promoted the growth of xenografts in nude mice (570 ± 132 mm³), while simultaneously transfected with EDPTOR overexpressing (169 ± 65 mm³) almost totally abrogated EGFR-enhanced tumor growth (Fig. 7B), as well as tumor weights (Fig. 7C). Taken together, these in vivo tumor growth results were in agreement with our in vitro results and indicated that DEPTOR is a pivotal tumor suppressor that was downregulated by the EGFR signaling pathway to promote tumor growth of lung adenocarcinoma.

The EGFR signaling pathway plays a critical role in promoting cancer cell growth and survival, which serves as an attractive therapeutic target, is attracting even more and more attention (49). EGFR tyrosine kinase inhibitors (TKI), such as gefitinib, which operate by repression of EGFR oncogenic signaling, have proven to provide a significant response and survival benefit for patients with lung cancer. However, all responders eventually acquire chemotherapy resistance (15, 50), and lung cancer is still estimated to be responsible for nearly one in five (1.59 million deaths, 19.4% of the total) of death from cancer worldwide (http://globocan.iarc.fr/Default.aspx). Because we found DEPTOR upregulated in response to gefitnib treatment (Fig. 3B), it would be interesting to test the role of DEPTOR in gefitnib resistance, the exact molecular mechanisms of which we are still unclear about.

One of the downstream pathways controlled by EGFR involves mTOR, a proto-oncogene activated in various cancers (51). The kinase mTOR is a critical target of EGFR signaling, linking growth factor abundance to cell growth and proliferation (52). DEPTOR, as a natural negative regulator of mTOR, has been reported to involve in sensitive to apoptosis gene (SAG)-induced lung tumor formation and may inhibit cell growth of lung adenocarcinoma (34); furthermore, loss of DEPTOR increased drug resistance to EGFR TKIs of lung adenocarcinoma cells (53). Thus, we questioned whether DEPTOR serves as a tumor suppressor and EGFR downregulates DEPTOR expression in lung adenocarcinoma.

To answer this question, we first detected the mRNA expression of EGFR and DEPTOR in clinical samples from patients with lung adenocarcinoma. Our results showed that EGFR expression increases with TNM stage and lymph node metastasis, while DEPTOR shows a contrary tendency (Table 1), and there is significant negative correlation between EGFR and DEPTOR expression (Fig. 1). In the EGFR signaling cascade, ligand stimulation promotes EGFR phosphorylation and then leads to receptor internalization and downstream signaling activation (54). In lung adenocarcinoma patients, we also observed that EGFR/pEGFR is partially expressed in the cytoplasm that DEPTOR mainly located (Fig. 2A).

Next, we chose human lung adenocarcinoma A549 cell lines to conduct our study and cloned EGFR overexpressing A549 cells. Compared with parental cells, EGFR ectopic expression significantly decreased DEPTOR expression; this could be further enhanced with the presence of EGF, EGFR ligand. More importantly, DEPTOR expression was enhanced with the stimulation of gefitinib. The following molecular analysis further identified that EGFR activation enhanced mTOR and S6K phosphorylation, CK1α and βTrCP1 expression. Of these four molecules, S6K is a direct downstream molecule of mTOR (55) and consists of the autoamplification loop of mTOR to degrade DEPTOR expression (27, 30). CK1α and βTrCP1 are involved in promoting tumor progression and have been found to act synergistically with EGFR to inhibit cell expansion of lung cancer (56, 57), but how EGFR activation stimulates CK1α and βTrCP1 expression is still unknown and needs to be identified in our future study.

The role of DEPTOR expression in the progression of human malignancies is still controversial; this may be because the regulation of DEPTOR is complicated in different individuals and tissues (28, 31), as two opposite results were obtained in the same myeloma cells (31, 58). In lung adenocarcinoma, there is still no direct evidence that DEPTOR regulates tumor progression. By constructing two stable A549 cell lines, namely, DEPTOR overexpression or knockout, we first demonstrated that DEPTOR acts as a tumor suppressor in the tumorigenesis of lung adenocarcinoma, as DEPTOR inhibited cell proliferation, migration, invasion, and in vivo tumor growth. Furthermore, simultaneous knockin DEPTOR in EGFR overexpressing A549 cells suppressed EGFR expression and markedly abolished EGFR-driven tumorigenesis of lung adenocarcinoma. Accompanied with the observation that DEPTOR expression provides a feedback promotion on EGFR protein degradation, more detailed molecular mechanisms need to be investigated in the future.

Of course, there are still some potential problems with our current study. For example, it is well known that CRISPR/Cas9-mediated gene editing has off-target effects. Although we believe that the guide RNA sequence has been tested by the commercial provider for our study, there might still be chances that our guide RNA can target non-DEPTOR sequence in human genome. We will take this into consideration in future studies. Inspired by two elegant studies from leading scientists in the CRISPR/Cas9 research field (59, 60), we will try to use modified Cas9 nucleases with high fidelity in our future studies, with the hope of minimal off-target effects.

In conclusion, our study reveals a novel link between EGFR and DEPTOR, and that EGFR promotes DEPTOR degradation by enhancing the function of the mTOR autoamplification loop, including increased mTOR and S6K phosphorylation, as well as CK1α and βTrCP1 expression. Furthermore, we also provide solid experimental evidence that DEPTOR plays a tumor suppressive role in lung adenocarcinoma cells, and ectopic expression of DEPTOR could suppress EGFR-driven tumor progression.

No potential conflicts of interest were disclosed.

Conception and design: X. Zhou, J. Guo, H. Zhu, J. Zhao

Development of methodology: X. Zhou, J. Guo, H. Zhu

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): X. Zhou, J. Guo, G. Pan, T. Liu

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): X. Zhou, J. Guo, Y. Ji, H. Zhu, J. Zhao

Writing, review, and/or revision of the manuscript: X. Zhou, J. Guo, Y. Ji, H. Zhu, J. Zhao

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): X. Zhou, J. Guo, G. Pan, T. Liu, J. Zhao

Study supervision: X. Zhou, J. Guo, J. Zhao

This work was supported by the National Natural Science Foundation of China (grant no. 81101775) and American Heart Association Grant 12SDG12070174.

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.