Lung cancer is the leading cause of cancer-related mortality worldwide. The mortality is high mainly due to the lack of known effective screening procedures; there is a high tendency for early spread and systemic therapies do not cure metastatic disease. Thus, it is important to investigate the molecular mechanism(s) of lung cancer development and, specifically, to identify an effective method by which to inhibit the invasion and metastasis of lung cancer. Ubiquitin-conjugating enzyme 9 (Ubc9), the sole conjugating enzyme for sumoylation, regulates protein function and plays a key role in tumorigenesis. Whether Ubc9 is involved in the invasion and metastasis of lung cancer remains unknown. Herein, we report that Ubc9 exhibits an important role in lung cancer invasion and metastasis. We first investigated the biological effect of Ubc9 on lung cancer by cloning the Ubc9 gene into a eukaryotic expression plasmid and stably expressing it in the human small cell lung cancer cell line NCI-H446 in order to observe any biological changes. We further analyzed the effect of Ubc9 in an
Despite the significant progress toward the understanding of human lung cancer tumorigenesis in the past two decades, lung cancer remains the leading cause of cancer-related mortality worldwide (
Increasing evidence demonstrates that sumoylation is a multistep process analogous to the ubiquitin pathway and involves maturation, activation, conjugation, ligation, and deconjugation steps. Sumoylation, which includes small ubiquitin-like modifier (SUMO) protein addition or removal from other proteins, is a post-translational modification that is significantly involved in diverse cellular processes, including transcriptional regulation, nuclear transport, cell cycle control, and maintenance of genome integrity through modulating protein-protein interactions of target proteins (
In the present study, we report higher levels of Ubc9 expression in primary lung cancer and metastatic nodules than in premalignant and/or normal tissues. Furthermore, we observed that inducing upregulation of Ubc9 expression in lung cancer cells promotes migration and invasion. Based on these findings, we concluded that Ubc9 may play an important role in cancer progression and that Ubc9 promotes invasion and metastasis in lung cancer.
The lung cancer cell lines A549, NCI-H460, NCI-H446, NCI-H292 were obtained from the American Type Culture Collection. The MCF-7 cell line was a gift from the Department of Cell Biology, China Medical University. Cell culture reagents were obtained from Gibco BRL (Grand Island, NY, USA). Protease inhibitors (leupeptin, aprotinin, Na3VO4, and phenylmethylsulfonylfluoride) were purchased from Sigma Aldrich (St. Louis, MO, USA). Goat anti-human Ubc9 antibody and GAPDH antibody were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Horseradish peroxidase (HRP)-conjugated secondary antibody was obtained from Santa Cruz Biotechnology, Inc. Reagents for SDS-PAGE were obtained from Bio-Rad (Hercules, CA, USA). Chemiluminescent developing reagents and a Micro BCA Protein Assay Reagent kit were obtained from Pierce Chemical Co. (Rockford, IL, USA). All other reagents were of analytical grades.
Tissue samples were obtained from a total of 143 patients with lung cancer who were treated at Liaoning Cancer Hospital and Institute from 2006 to 2010. Institutional Review Board approval was obtained to procure and analyze the tissues used in this study. None of the patients had received preoperative neo-adjuvant chemotherapy or radiation therapy. Histological types included squamous carcinoma, adenocarcinoma and small cell lung cancer (SCLC) (
All specimens for immunohistochemical staining were fixed in 10% neutral formalin, embedded in paraffin and cut in 5-μm serial sections. Immunohistochemical staining was performed using a peroxidase-labeled streptavidin-biotin technique. Briefly, tissue sections were deparaffinized and rehydrated. Then, sections were heated in a microwave oven for 10 min to retrieve antigenicity and were treated with 3% H2O2 in methanol for 10 min to quench endogenous peroxidase activity. After washing in 10 mM PBS (pH 7.6), sections were incubated with 10% normal goat serum (Solarbio Science and Technology, Beijing, China) for 10 min to block nonspecific antibody binding. Sections were then incubated overnight at 4°C with goat anti-human Ubc9 polyclonal antibody (1:100). After washing in PBS, sections were treated with a 1:100 dilution of biotinylated donkey anti-goat IgG for 30 min followed by a streptavidin-peroxidase conjugate for 30 min (Dako). A solution of 0.02% diaminobenzidine hydrochloride (DAB) containing 0.03% H2O2 was used as chromogen to visualize peroxidase activity. The preparations were lightly counterstained with hematoxylin, mounted with Permount (Thermo Fisher Scientific, Waltham, MA, USA) and examined by light microscopy.
Human Ubc9 cDNA (GenBank accession no. NM_003345) was obtained by reverse transcription from NCI-H446 cells using the following primers: 5′-CGGAATTCCCACCATGTCGGGGAT-3′ and 5′-CGGGATCCAATGAGGGCGCAAAC-3′. PCR conditions were: 95°C for 2 min (94°C 10 sec, 58°C 1 min, 72°C 2.5 min) 30 cycles at 72°C for 2 min. The PCR product was cloned into the pGEM-T vector (Promega, Madison, WI, USA). The resulting plasmid, pGEM-T-Ubc9, was sequence-verified. The Ubc9 fragment was then sub-cloned into the pEGFP-vector (Invitrogen, Carlsbad, CA, USA) using the
The human lung cancer cells A549, NCI-H460, NCI-H446, NCI-H292 and MCF-7 were maintained in RPMI-1640 medium containing 10% FBS, 100 IU/ml penicillin and 100 μg/ml streptomycin. All cell lines were incubated in 5% CO2 at 37°C. Stable transfections of NCI-H446 cells were carried out with the following expression vectors: pEGFP-N1-vector and pEGFP-N1-Ubc9. Single cell clones were selected using G418 and confirmed by western blotting.
Western blotting was performed using standard techniques as previously described. Briefly, cells were washed twice with PBS buffer and lysed in RIPA lysis buffer (50 mM Tris-Cl pH 7.4, 150 mM NaCl, 0.5% sodium deoxycholate, 1% NP-40, 0.1% SDS, 1 mM EDTA, 100 mM NaF, 1 mM Na3VO4, 1 mM PMSF, and 2 μg/ml aprotinin) on ice. Total proteins (50 μg) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride membranes. Membranes were blocked with 5% nonfat milk in TBST (10 mM Tris, pH 7.4, 150 mM NaCl and 0.1% Tween-20) at room temperature for 2 h and incubated with the indicated primary antibodies at 4°C overnight with gentle rocking. After washing with TBST, the membrane was reacted with the appropriate HRP-conjugated secondary antibodies for 1 h at room temperature. After extensive washing with TBST, proteins were visualized by the enhanced chemiluminescence (ECL) detection kit in accordance with the manufacturer’s recommendations.
NCI-H446 cells (1.50×104) were placed in the upper chamber of 8 μm Transwells (Costar Inc.) on 100 μl of solid, growth factor reduced Matrigel (BD Biosciences, Bedford, MA, USA). RPMI-1640 medium supplemented with 20% FBS was added to the wells and then cells were incubated for 4 h at 37°C. At the end of the migration assay, the filter side of the upper chamber was cleansed with a cotton swab and the filter was stained for 1 h with crystal violet (Sigma) in 2% ethanol and rinsed with water. The filter was gently cut from the chamber and the cells that had migrated through the filter pores from the underside of the filter were counted. Four high-power fields per insert were counted and the values were averaged. Both for NCI-H446+pEGFP-N1-vector and NCI-H446+pEGFP-N1-Ubc9, three identical replicates were performed.
Cells were seeded in 6-well plates and cultured until confluent. A pipette tip was used to make a straight scratch across the diameter of the dish, simulating a wound. Cell migration was documented using phase contrast microscopy at ×10 at 12, 24, 48 and 72 h.
Nude mice were anesthetized by intraperitoneal injection of Nembutal [45 mg/kg of pentobarbital (50 mg/ml)/saline/ethanol/propylene glycol 10/63/7/18]. The anterior chest wall was scrubbed with 70% alcohol. A 30-gauge needle on a tuberculin syringe was inserted into the second intercostal space 3 mm to the left of the sternum and aimed centrally. The spontaneous and continuous entrance of pulsating blood into the transparent needle hub indicated proper positioning of the needle into the left ventricle of the heart. Tumor cells [106 cells in 0.1 ml Hank’s balanced salt solution (HBSS)] were injected over a 20–40 sec period.
Statistical analysis was performed using the Student’s t-test. Data are presented as the means ± SD and p-values <0.05 were considered to indicate statistically significant differences. All calculations were carried out using the SPSS13.0 statistical package.
We observed Ubc9 immunostaining in all human lung tissue sections and normal tissue. The Ubc9 protein level was markedly elevated in the lung tumor specimens compared with the normal lung tissue, including both non-small cell lung cancer (NSCLC) and SCLC tissue types. Moreover, the level of Ubc9 protein in NSCLC was higher than in SCLC (
The level of Ubc9 mRNA in lung cancer tissues was quantitated using real-time PCR. Ubc9 mRNA expression of Ubc9 was again detected in lung normal tissue and lung cancer tissue, including NSCLC and SCLC. Similar to the levels of Ubc9 protein, the level of Ubc9 mRNA in NSCLC samples was significantly higher than in SCLC samples (
The invasive ability of different lung cancer cells was analyzed using a cell invasion assay (
To further investigate the role of Ubc9 in lung cancer, we constructed the mammalian expression vector: pEGFP-N1-Ubc9 and pEGFP-N1-vector, stably transfected NCI-H446 cells, a SCLC cell, generated a stable cell line expressing pEGFP-N1-Ubc9 and pEGFP-N1-vector. Expression of the fusion protein in the pEGFP-N1-Ubc9 clones was detected by western blotting (
To directly test the hypothesis that Ubc9 has a causal role in the metastasis of lung cancer cells, we used stable transfection of Ubc9 cDNA to increase the level of Ubc9 protein in NCI-H446 cells. Then, we injected these cells into the left heart ventricle of nude mice in order to evaluate their ability to metastasize to the lung. Injection of either the pEGFP-N1-vector/NCI-H446 cells or the pEGFP-N1-Ubc9/NCI-H446 cells produced lung metastatic lesions in the injected mice. Some visible metastatic nodules were observed even in nude mice injected with either the pEGFP-N1 vector/NCI-H446 or the pEGFP-N1-Ubc9/NCI-H446 cells. However, the metastatic ability of pEGFP-N1-Ubc9/NCI-H446 cells was markedly increased compared with that of the control cells (
As is well known, post-translational modifications play an important role in protein function through the regulation of their activity, turnover and localization and/or interactions. One such modification involves the covalent attachment of the small ubiquitin-related polypeptide SUMO (small ubiquitin-like modifier) to different cellular protein substrates (
Ubc9 is the sole E2 conjugating enzyme required for protein sumoylation (
Ubc9 has been specifically linked to tumorigenesis. Ubc9 is overexpressed in several malignancies, such as lung adenocarcinoma (
In accordance with previous studies (
To further determine whether Ubc9 has a function in tumor metastasis, we performed
Metastasis is detected in 90% of lung cancer-related deaths (
In summary, we provided clinical and experimental evidence that Ubc9 is differentially expressed in lung cancer cells and contributes to lung cancer cell migration and metastasis. Ubc9 may serve as a potential biomarker as a therapeutic target for cancer intervention.
The study was supported by Outstanding Scientific Fund of Shengjing Hospital.
The expression of Ubc9 in lung cancer tissue. (A) The expression of Ubc9 in lung cancer tissues was detected using immunochemistry. (B) The results were analyzed by Image-Pro plus software. (C) The expression of Ubc9 mRNA in lung cancer tissue was detected using real-time PCR. *P<0.05 vs. control.
The expression of Ubc9 in lung cancer cells correlates with invasion ability. The expression of Ubc9 in A549, NCI-H460, NCI-H446, NCI-H292 and MCF-7 cells was detected by RT-PCR (A) and western blotting (B). (C) The invasion of lung cancer cells was analyzed by invasion assay. Representative results of three independent experiments are shown. Data represent means ± SD of three independent experiments.
Effect of Ubc9 overexpression in NCI-H446 cells on NCI-H446 cell invasion and migration. (A) NCI-H446 cells transfected with Ubc9 cDNA and pEGFP-N1 vector were designated as pEGFP-N1-Ubc9 and pEGFP-N1-vector, respectively. Ubc9 expression in transfected cells was analyzed by western blotting. (B) Invasion ability was analyzed using a Matrigel invasion assay; migration was analyzed using a cell scratch assay (C). Results representative of three independent experiments are shown. Data is presented as means ± SD. *P<0.05 vs. control.
The overexpression of Ubc9 in NCI-H446 cells promotes lung cancer cell metastasis to lung. (A) Growth patterns of tumors formed by NCI-H446+pEGFP-N1-vector cells or NCI-H446+pEGFP-N1-Ubc9 cells treated with intracardiac injection of tumor cells. (B) Histological analyses of lung metastatic tumors. Magnification, ×100.
Correlation between Ubc9 expression and clinical pathological factors.
Variable | Case (n and %) | The level of Ubc9 expression | χ2 value (P) | |
---|---|---|---|---|
|
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> median values (n) | < median values (n) | |||
Gender | ||||
Male | 97 (67.83%) | 51 | 46 | 2.26 (>0.05) |
Female | 46 (32.17%) | 18 | 28 | |
Age (years) | ||||
>60 | 51 (35.66%) | 40 | 11 | 25.01 (<0.01) |
≤60 | 92 (64.34%) | 32 | 60 | |
Histological type | ||||
Squamous carcinoma | 31 (21.68%) | 18 | 13 | 7.02 (>0.05) |
Adenocarcinoma | 28 (19.58%) | 9 | 19 | |
SCLC | 79 (55.25%) | 42 | 37 | |
Adenosquamous ca. | 1 (0.7%) | 1 | 0 | |
Large cell carcinoma | 1 (0.7%) | 0 | 1 | |
Alveolar carcinoma | 3 (1.09%) | 1 | 2 | |
Grade of differentiation | ||||
Poor | 95 (66.43%) | 50 | 45 | 0.45 (>0.05) |
Moderate | 32 (22.38%) | 16 | 16 | |
Well | 16 (11.19%) | 7 | 9 | |
Nodal status | ||||
N (+) | 118 (82.52%) | 78 | 40 | 5.94 (<0.05) |
N (−) | 25 (17.48%) | 10 | 15 | |
TNM | ||||
I | 41 (28.67%) | 15 | 26 | 9.57 (<0.05) |
II | 32 (20.98%) | 20 | 12 | |
III | 50 (34.97%) | 28 | 22 | |
IV | 20 (15.38%) | 15 | 5 | |
Survival | ||||
Yes | 91 (63.64%) | 33 | 58 | 16.12 (<0.01) |
No | 52 (36.36%) | 37 | 15 |
Effects of upregulation of Ubc9 expression on lung metastasis in nude mice.
Lung metastasis | |||
---|---|---|---|
| |||
Cell line | Incidence | Survival | |
NCI-H446 cells | HBSS | 0/4 | All >90 |
pEGFP-N1-vector | 3/10 | 56 to >90 | |
pEGFP-N1-Ubc9 | 8/10 | 40 to >90 |