Introduction
Lung cancer arises from the bronchial mucosal epithelium and it is the leading cause of cancer mortality worldwide. Non-small cell lung cancer (NSCLC) is the most commonly diagnosed type of lung cancer, accounting for approximately 85% of all cases. Although the continuous progress has been made for surgical resection, chemotherapy, and radiation therapy [
1-
3], prognoses have not significant improved. In recent years, molecular targeted therapy [
4,
5] has become the most prevalent approach. Therefore, the understanding of the molecular alterations in NSCLC and their pathways is significant for molecular targeted therapy.
During tumor formation and expansion, increasing glucose metabolism is necessary for the unrestricted growth of tumor cells [
6]. Distributed in a variety of tissues, α-enolase (ENO1) was originally described as an enzyme responsible for the glycolytic pathway [
7]. In addition to its glycolytic function, accumulating evidence has demonstrated that ENO1 is a multifunctional protein involved in several biological and pathophysiological processes depending on its cellular localization [
8]. The molecular weight of ENO1 protein is 48 kDa. It is expressed in the cytoplasm and considered as an oncogene in tumor pathogenesis. However, another transcript of ENO1 can be translated into a 37-kDa c-Myc promoter-binding protein (MBP-1), which represses transcription and is localized in the nucleus [
9-
11].
Overexpression of ENO1 has been previously demonstrated in several types of tumors including NSCLC [
12]. However, investigators have reported conflicting results. Some researchers have shown that the expression of ENO1 was upregulated in NSCLC tissues and was associated with poorer clinical outcomes [
13,
14]. On the contrary, Chang Y.S.
et al. demonstrated that the levels of ENO1 protein were significantly decreased in NSCLC [
15] and overexpression of ENO1 inhibited epithelial-mesenchymal transition (EMT) in the A549 cell line [
16]. Therefore, neither expression nor the functional mechanisms of ENO1 in NSCLC have been clearly established.
In order to further validate the role of ENO1 and its molecular basis in NSCLC, we analyzed the expression of ENO1 in human NSCLC tissues and cell lines, as well as its effects on cell glycolysis, growth, migration, and invasion
in vitro and tumorigenicity and metastasis
in vivo. Our study showed that ENO1 is overexpressed in NSCLC tissues, and upregulated ENO1 promotes cell glycolysis, proliferation, migration, invasion, and tumorigenicity via the FAK/PI3K/AKT pathway. This is the first report of the molecular mechanisms of ENO1 in NSCLC, even more in-depth than our previous report of ENO1 in glioma [
17].
Discussion
Upregulated expression of ENO1 has been detected in several cancers, such as glioblastoma [
20], head and neck cancer [
21], pancreatic cancer [
22], and prostate cancer [
23]. However, the role of ENO1 in NSCLC is still controversial [
13-
16], which needs to be further identified. In this study, we confirmed that the expression of ENO1 mRNA and protein was frequently overexpressed in NSCLC tissues compared to non-cancerous lung tissues as well as in NSCLC cells compared to HBE cells. These results are consistent with Chang
et al.’s report supporting an oncogenic role for ENO1 in NSCLC [
13], but not Chang’s study [
15].
In order to evaluate the function of ENO1 and eliminate the influence of MBP-1 on NSCLC, we firstly performed an immunofluorescence and observed that ENO1 was expressed in the cytoplasm but not in the nucleus (MBP-1) in A549 and SPCA-1 cells. Furthermore, we also found that MBP-1 was not expressed by Western blot assay in A549 and SPCA-1 cells. The abovementioned results suggested that both two cells could be used as well-defined models to evaluate the function of ENO1 on NSCLC. Further, stable ENO1-overexpressed A549 cells and stable ENO1-suppressed SPCA-1 cells as well as transient ENO1-suppressed A549 and SPCA-1 cells were respectively constructed, which was used to investigate the role of ENO1 in NSCLC.
ENO1 was originally described as an enzyme responsible for the glycolytic pathway. To further assess the effect of ENO1 on NSCLC cells, we analyzed the glycolysis changes triggered by ENO1 and found that overexpressed and suppressed ENO1 respectively increased and decreased the production of lactate. These data suggested that ENO1 was involved in inducing glycolysis in NSCLC.
Previous studies have demonstrated that ENO1 overexpression was positively associated with progression and poor prognosis in neuroendocrine tumors, neuroblastoma, pancreatic cancer, prostate cancer, cholangiocarcinoma, thyroid carcinoma, hepatocellular carcinoma, and breast cancer [
14,
21-
27]. Further, ENO1 has been shown to promote cell proliferation, cycle progression, migration, and invasion [
14,
21-
33], which suggests that ENO1 functions as an oncogene in tumor pathogenesis. In this study, we found that overexpressed ENO1 significantly elevated cell proliferation and clone formation
in vitro as well as tumorigenesis
in vivo. Furthermore, we also observed that overexpressed ENO1 induced cell migration, invasion, and metastasis in NSCLC. Our results are consistent with previous reports in other tumors that ENO1 functions as an oncogene [
13,
14,
29,
34] but are in contrast with Zhou
et al.’s report that ENO1 overexpression suppressed EMT in NSCLC A549 cell line [
16].
The biological functions of ENO1 found in this study provide a mechanistic basis for the pathological and clinical observations. When we examined the key regulators of the glycolysis and cell cycle at the G1-S phase transition, we discovered that suppression of ENO1 inhibited the expression of LDHA, c-Myc, cyclin D1, p-Rb, and cyclin E1 while elevating the expression of p21, which promoted cell glycolysis and proliferation of NSCLC. EMT is regarded as a key event in tumor migration and invasion progression. In this study, we further examined the expression of EMT marker genes and found that knocking down ENO1 expression induced the protein levels of E-cadherin while suppressing the expression of snail, vimentin, and N-cadherin in NSCLC cells. These results are consistent with our previous report of ENO1 in glioma [
17]. However, ENO1 overexpression did not lead to any changes from epithelial to mesenchymal transition in NSCLC cells.
PI3K/AKT is a key signal mediator during carcinogenesis [
35,
36], and its activation induces glycolysis [
37-
39] and c-Myc-mediated cell cycle transition [
40] and promotes the progression of EMT [
37,
41]. In addition, c-Myc has also been shown to regulate energy metabolism by regulating LDHA in tumor [
42]. We hypothesized that oncogenic ENO1 functions through the PI3K/AKT pathway in NSCLC. We found that suppressed ENO1 significantly decreased the protein levels of β-catenin and phosphorylated PI3K and AKT, but not their total protein levels in SPCA-1 cells, which is similar to our previous report in glioma [
17]. Interestingly, we examined the protein levels of FAK, an upstream signal factor of the PI3K/AKT pathway, and found that suppressed ENO1 significantly decreased levels of phosphorylated FAK, but not its total protein levels. We speculated that ENO1 regulates cell glycolysis, proliferation, migration, and invasion through FAK-mediated PI3K/AKT pathway in NSCLC. To further clarify the specific mechanism, Ang II, an activating agent of phosphorylated FAK [
19], was used to treat ENO1-suppressed SPCA-1 cells. We observed that not only the production of lactate, cell viability, migration, and invasion was restored but also the expression levels of p-FAK, p-AKT, LDHA, cyclin D1, c-Myc, p21, and β-catenin were rescued. These results demonstrated that suppressed ENO1 inhibited cell glycolysis, proliferation, migration, and invasion by inactivating FAK-mediated PI3K/AKT pathway in NSCLC. Thus, ENO1 may be a potential therapeutic target for NSCLC treatment. Furthermore, nanotechnology has provided a good platform for cancer targeted therapy based on nanoparticle unique properties. Therefore, we wish to develop a nanoparticle formulation modified with tumor-targeting single-chain antibody fragment (scFv) for systemic delivery of siRNA-ENO1 in the future [
43], which may make it possible that ENO1 serves as a molecular therapeutic target for NSCLC treatment.
Materials and methods
Cell culture and sample collection
A549 line was obtained from ATCC Bioresource Center, and SPCA-1 and HBE were purchased from Chinese Academy of Sciences Cell Bank (Shanghai, China). A549 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, HyClone, Logan, UT) supplemented with 10% fetal bovine serum (FBS) (ExCell, Shanghai, China); SPCA-1 was cultured in RPMI 1640 medium (HyClone, Logan, UT) supplemented with 10% FBS (ExCell, Shanghai, China). HBE, an immortalized human bronchial epithelial cell line, was grown in DMEM (HyClone, Logan, UT) supplemented with 20% FBS (ExCell, Shanghai, China). All cell lines were maintained in a humidified chamber with 5% CO2 at 37°C. Twenty-six (26) surgical resected fresh primary NSCLC tissues and paired para-cancer lung tissues as well as non-cancerous lung tissues (5 cm away from tumor edge), 55 paraffin-embedded primary NSCLC specimens, and 17 paraffin-embedded non-cancerous lung specimens were obtained from the Third Affiliated Hospital of Kunming Medical University (Yunnan, China). Patients with a diagnosis of relapse and who had received preoperative radiation, chemotherapy, or biotherapy were excluded from the study to avoid any changes in tumor marker determination due to the effect of the treatment. The clinical processes were approved by the Ethics Committees of the Third Affiliated Hospital of Kunming Medical University, and patients provided informed consent. Demographic and clinical data were obtained from the patients’ medical records.
RNA isolation, RT-PCR, qRT-PCR, and primers
Total RNA was extracted from the cell lines and lung tissues using Trizol (Takara, Shiga, Japan). RNA (1 μg) was reverse transcribed into cDNA, and cDNA was used as a template to amplify with specific primers for sense: 5′-TCAATGGCGGTTCTCATGCT-3′ and for antisense: 5′-GCAGCTCCAGGCCTTCTTTA-3′. ARF5 was used as an internal control with primers for sense: 5′-ATCTGTTTCACAGTCTGGGACG-3′ and for antisense: 5′-CCTGCTTGTTGGCAAATACC-3′. Experiments were performed according to the manufacturer’s instructions (Takara, Shiga, Japan). PCR conditions were 95°C for 10 min to activate DNA polymerase, followed by 45 cycles of 95°C for 15 s, 60°C for 15 s, and 72°C for 15 s. Specificity of amplification products was determined by melting curve analysis. The qRT-PCR reactions for each sample were repeated three times. Independent experiments were done in triplicate.
Immunohistochemistry and evaluation of staining
Immunohistochemistry was performed in primary NSCLC tissues, non-cancerous lung tissues, and mouse tumors according to a previous description [
44] with rabbit polyclonal anti-ENO1 antibody (1:150; Proteintech, USA), anti-cyclin D1, p21 antibody (1:100; Epitomics, Burlingame, USA), β-catenin, N-cadherin, or E-cadherin antibody (1:100; Cell Signaling Technology, Danvers, USA).
Stained tissue sections were reviewed and scored independently by two investigators blinded to the clinical data. For cytoplasmic staining, the score was based on the sum of cytoplasm staining intensity and the percentage of stained cells. The staining intensity was scored as previously described (0–3) [
44,
45], and the percentage of positive staining areas of cells was defined as a scale of 0–3 (0: <10%, 1: 10%–25%, 2: 26%–75%, and 3: >76%). For nuclear staining, the staining score was defined based on the sum of nuclear staining intensity and the percentage of positive nuclear staining. Positive nuclear staining scores were defined as follows: 0: <20%, 1: 20%–49%, 2: 50%–79%, and 3: >80%. The sum of the staining intensity and staining extent scores (0–6) was used as the final staining score. For statistical analysis, a final staining score of 0 ~ 2 and 3 ~ 6 in cytoplasm or 0 ~ 3 and 4 ~ 6 in nucleus were respectively considered to be negative and positive expression levels. Expression of ENO1 in the nucleus was observed, but since ENO1 localizes in cytoplasm, only the cytoplasmic staining was evaluated.
Immunofluorescence
Immunofluorescence was performed according to a previous study [
46]. NSCLC cells were seeded on coverslips in six-well plate and cultured overnight. Subsequently, cells were fixed in 3.5% paraformaldehyde and permeabilized in KB solution and 0.2% Triton X-100 at room temperature. After the blocking solution was washed out, cells were incubated with a primary antibody (ENO1) (diluted in KB) for 30–45 min at 37°C and subsequently washed with KB twice. After incubating for 30–45 min at 37°C with secondary antibody (diluted in KB) and washing with KB again, the coverslips were then mounted onto slides with mounting solution containing 0.2 mg/ml DAPI and sealed with nail polish. Slides were stored in a dark box and observed under a fluorescent microscope.
Western blot analysis, reagent, and antibodies
Western blotting was performed as described [
47] with rabbit polyclonal anti-ENO1, LDHA antibody (1:1,000; Proteintech, USA), anti-cyclin D1, p21, cyclin E1, C-Myc antibody (1:1,000; Epitomics, Burlingame, USA), anti-CDK4 antibody (1:400; Santa Cruz Biotechnology, Santa Cruz, USA), anti-pRb (Ser780), FAK, p-FAK (Tyr397), AKT, p-AKT (Ser473), PI3K, p-PI3K (Tyr458), snail, β-catenin, N-cadherin, vimentin, and E-cadherin antibody (1:1,000; Cell Signaling Technology, Danvers, USA). An HRP-conjugated anti-rabbit IgG antibody was used as the secondary antibody (Zhongshan, Beijing, China). Signals were detected using enhanced chemiluminescence reagents (Pierce, Rockford, IL). Ang II was purchased from the Santa Cruz Biotechnology (Santa Cruz, USA).
Transfection and infection
The full-length ENO1-GFP (ENO1), GFP empty vector (PLV-Ctr) lentiviruses were designed by Shanghai Genechem (Genechem, Shanghai, China). The preparation of lentiviruses expressing human ENO1 short hairpin RNA (shENO1-A, B, C) (Table
2) was performed using the pLVTHM-GFP lentiviral RNAi expression system [
40]. NSCLC cell line A549 was infected with full-length ENO1-GFP or GFP empty vector lentiviruses. SPCA-1 cells were infected with shENO1-A, B, C or PLV-shCtr lentiviruses, and polyclonal cells with GFP signals were selected for further experiments using FACS flow cytometry. Total RNA was isolated, and levels of ENO1 mRNA were measured using real-time PCR analysis.
Table 2
shRNA sequences for ENO1
A | Sense | 5′-CCGGAATGTCATCAAGGAGAAATATCTCGAGATATTTCTCCTTGATGACATTTTTTTG-3′ |
Antisense | 5′-AATTCAAAAAAATGTCATCAAGGAGAAATATCTCGAGATATTTCTCCTTGATGACATT-3′ |
B | Sense | 5′-CCGGCGTGAACGAGAAGTCCTGCAACTCGAGTTGCAGGACTTCTCGTTCACGTTTTG-3′ |
Antisense | 5′-AATTCAAAACGTGAACGAGAAGTCCTGCAACTCGAGTTGCAGGACTTCTCGTTCACG-3′ |
C | Sense | 5′-CCGGCCACTGTTGAGGTTGATCTCTCTCGAGAGAGATCAACCTCAACAGTGGTTTTTG-3′ |
Antisense | 5′-AATTCAAAAACCACTGTTGAGGTTGATCTCTCTCGAGAGAGATCAACCTCAACAGTGG-3′ |
Transient transfection with siRNAs
siRNA for ENO1 was designed and synthesized by Guangzhou RiboBio (RiboBio Inc, China). The sequence of siENO1 is sense: 5′-GCAUUGGAGCAGAGGUUUAdTdT-3′ and anti-sense: 3′-dTdTCGUAACCUCGUCUCCAAAU-5′. The sequence of si-negative control (si-Ctr) was also designed by RiboBio (RiboBio Inc, China). Twenty-four hours prior to transfection, NSCLC cells A549 and SPCA-1 were plated onto a 6-well plate or a 96-well plate (Nest Biotech, China) at 30%–50% confluence. They were then transfected into cells using TurboFect TM siRNA Transfection Reagent (Fermentas, Vilnius, Lithuania) according to the manufacturer’s protocol. Cells were collected after 48–72 h for further experiments.
Metabolic profiles were obtained to assess the relative distribution of various cellular metabolites of NSCLC cells. Cells were collected and quickly frozen. Further sample preparation, metabolic profiling, peak identification, and curation were performed by Metabolon (Durham, NC, USA) using their described methods [
48].
MTT assay
The viability of cell proliferation was assessed using MTT assay according to our previous study [
46]. Cells were seeded in 96-well plates at a density of 1,000 cells/well. Every 24 h for 7 days, 20 μl of MTT (5 mg/ml) (Sigma-Aldrich, St. Louis, MO) was added to each well and incubated for 4 h. Supernatants were removed, and 150 μl of dimethyl sulfoxide (DMSO) (Sigma-Aldrich, St. Louis, MO) was added to each well. The absorbance value (OD) of each well was measured at 490 nm. For each experimental condition, five parallel wells were assigned to each group. Experiments were performed thrice.
Clone formation assay was performed according to our previous study [
46]. Cells were seeded in 6-well culture plates at 100 cells/well. Each cell group had three parallel wells. After incubation for 14 days at 37°C, cells were washed twice with Hank’s solution and stained with hematoxylin solution. The number of colonies containing ≥50 cells was counted under a microscope. The clone formation efficiency was calculated as (number of colonies/number of cells inoculated) × 100%.
Cell migration and invasion assays
In vitro cell migration and invasion assays were examined according to our previous study [
46]. For cell migration assays, 1 × 10
5 cells in a 100-μl medium without serum were seeded on a fibronectin-coated polycarbonate membrane insert in a transwell apparatus (Corning, USA). In the lower surface, 500 μl DMEM with 10% FBS was added as chemoattractant. After the cells were incubated for 10 h at 37°C in a 5% CO
2 atmosphere, Giemsa-stained cells adhering to the lower surface were counted under a microscope in five predetermined fields (100×). All assays were independently repeated at least thrice. For cell invasion assays, the procedure was similar to the cell migration assay, except that the transwell membranes were pre-coated with 24 μg/ml Matrigel (R&D Systems, USA).
In vivo tumorigenesis in nude mice
According to our previous study [
17], a total of 1 × 10
6 logarithmically growing A549 cells transfected with full-length ENO1 and PLV-Ctr vector, SPCA-1 cells transfected with shENO1-B, and the control PLV-shCtr vector (
N = 6 per group) in 0.1 ml Hank’s solution were subcutaneously inoculated into the left-right symmetric flank of 4–6-week-old male BALB/c-nu/nu mice. The mice were maintained in a barrier facility on HEPA-filtered racks and fed an autoclaved laboratory rodent diet. All animal studies were conducted in accordance with the principles and procedures outlined in the National Institutes of Health Guide for the Care and Use of Animals under assurance number A3873-1. After 15 days, the mice were sacrificed, and their tumors were excised, weighed, and processed for histology.
In vivo metastasis assays were performed according to a previous study [
46]. A total of 5 × 10
6 cells were injected into nude mice (
n = 5 for each group) through the spleen, respectively. The optical fluorescence images were visualized to monitor primary tumor growth and formation of metastatic lesions. Forty days later, all mice were killed, individual organs were removed, and metastatic tissues were analyzed by H&E staining.
Statistical analysis
All data were independently repeated at least thrice. SPSS 13.0 and Graph Pad Prism 5.0 software were used for statistical analysis. One-way ANOVA or two-tailed Student’s t-test were applied to determine the differences between group in vitro analyses. The chi-squared test was used to determine the differences of ENO1 protein expression between NSCLC tissues and non-cancerous lung tissues of the lung. A p value of less than 0.05 was considered statistically significant.
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Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
QFF, YL, YF, SNH, HYQ, and SWD performed the research; XS, WYF, and ZL designed the research study; RLL, YZ, XLY, MYZ, XJD, and YYC performed the statistical analysis; and QFF, RCL, RL, LBL, and WYF wrote the paper. All authors have read and approved the final manuscript.