Id-1 promotes migration and invasion of non-small cell lung cancer cells through activating NF-κB signaling pathway
© The Author(s). 2017
Received: 11 August 2017
Accepted: 1 December 2017
Published: 12 December 2017
Numerous studies have shown that Id-1 (Inhibitor of differentiation 1) is upregulated in several cancers and associated with tumor malignant characters. However, the clinical significance and biological role of Id-1 in non-small cell lung cancer (NSCLC) remains unclear.
We used RT-PCR, Western blot and Immunohistochemistry to measure Id-1 expression in NSCLC tissues and matched adjacent noncancerous tissues. The expression pattern of Id-1 in NSCLC tissues was determined by scoring system of immunohistochemical analysis. The Kaplan-Meier method was used to calculate the survival curve, and log-rank test to determine statistical significance. The Id-1 gene was overexpressed or downreuglated with Lentiviral vectors in NSCLC cells. And, the migration ability of NSCLC cells was tested in a Transwell Boyden Chamber.
We found that Id-1 is generally expressed higher in NSCLC tissues compared with matched adjacent noncancerous tissues. We also found that high Id-1 expression in tumor tissues is significantly correlated with tumor progression and poor survival in NSCLC patients. Furthermore, our experimental data revealed that knockdown of Id-1 significantly suppressed the proliferation, migration and invasion of NSCLC cells, whereas ectopic expression of Id-1 promoted the malignant phenotype of NSCLC cells. Mechanistic study showed that NF-κB signaling pathway contributed to the effects of Id-1 in NSCLC cells. Moreover, blocking the NF-κB pathway significantly inhibited the tumor-promoting actions of Id-1 in NSCLC cells.
We identified a tumorigenic role of Id-1 in NSCLC and provided a novel therapeutic target for NSCLC patients.
Lung cancer, predominantly non-small cell lung cancer (NSCLC) is the most commonly diagnosed malignancy, accounting for ~20% of all cancer-related death worldwide . While remarkable progress during the past decades in diagnose and treatment of NSCLC, but the predicted 5-year survival rate is only around 15% . And it is mainly a consequence of regional recurrence and lymph node metastasis [2, 12]. Thus, a better understanding of the molecular mechanism involved in the development and progression of NSCLC is urgently needed.
Inhibitor of differentiation 1 (Id-1), a class of dominant-negative antagonists of helix-loop-helix transcription factors, has multiple functions including inhibition of differentiation, induction of proliferation, and delaying replicative senescence [22, 29]. Recently, Id-1 has been suggested as a potential oncogene, as aberrant elevation of Id-1 has been found in numerous types of cancers such as cervical , osteosarcoma  and prostate cancers . In osteosarcoma and cervical cancer patients, high Id-1 expression is correlated with more aggressive behavior as well as much shorter overall survival [9, 24]. Furthermore, Id-1 has been reported to promote survival and metastatic ability of cancer cells. For instance, Id-1 enhances invasive properties of hematopoietic cell lines through transactivation of MMP9 . In addition, Id-1-induced transforming growth factor-β-induced epithelial–mesenchymal transition , and also enhanced cell migration in breast cancer . Although Id-1 has been extensively investigated in recent years, the functions and mechanisms of Id-1 in NSCLC development remain unclear.
In the present study, we found that Id-1 overexpression is significantly correlated with tumor progression and poor survival in NSCLC patients. Gain and loss of function assays found that Id-1 promoted migration, invasion and viability of NSCLC cells by in vitro experiments. Mechanistic study showed that the tumorigenic role of Id-1 in NSCLC is at least partly via activation of NF-κB signaling pathway. Therefore, our results identify Id-1 as a novel regulator of NSCLC invasiveness and may provide a promising therapeutic strategy for treatment of NSCLC patients.
Patients and tumor specimens
Matched cancerous and noncancerous tissues were obtained from 96 patients with NSCLC in the Department of General surgery, The General Hospital of The People’s Liberation Army, immediately snap-frozen in liquid nitrogen, and stored at −80 °C for RNA and protein extraction. A pathologist confirmed that all the specimens were derived from normal tissues. Informed consent was obtained from the patients, and the research procedure was approved by the Ethics Committee of The General Hospital of The People’s Liberation Army.
Cell lines and cell culture
A total of five cell lines were used including four NSCLC cell lines (H226, H292, H460 and A549) and the immortalized normal human bronchial epithelial cell line (BEAS-2B). NSCLC cells were maintained in RPMI-1640 (HyClone) supplemented with 10% FBS (Gibco) and antibiotics (100 U/ml penicillin and 100 mg/ml streptomycin) (Invitrogen). BEAS-2B cells were maintained in BEGM (Lonza). All cell lines were maintained in an incubator with a humidified atmosphere of 95% air and 5% CO2 at 37 °C.
RNA extraction and quantitative real-time PCR (qRT-PCR)
The relative RNA levels of genes were assessed by quantitative real-time PCR. In brief, total RNA was isolated with the standard TRIzol-based protocol (Invitrogen, USA). RNA was reverse transcribed using the PrimeScript RT Reagent Kit (Invitrogen, USA) and qPCR was performed using SYBR Premix Ex Taq (TaKaRa, China), following the manufacturer’s instructions. The gene-specific primers were as follows: GAPDH (sense: 5’-CTGGGCTACACTGAGCACC-3′, antisense: 5’-AAGTGGTCGTTGAGGGCAATG-3′), Id-1 (sense: 5’-CCAGCACGTCATCGACTAC-3′, antisense: 5′- GCTTCAGCGACACAAGATG-3′).
Western blot assay
Western blot analysis for specific protein expression was performed as previously described , while, cytoplasmic and nuclear protein were isolated and purified using the Protein and RNA Isolation System (Life technologies) according to the manufacturer’s instructions. The antibodies used were as follows: anti-Id-1 (1:500, Santa Cruz, USA), anti-p-IκBα (1:1000, CST), anti-IκBα (1:1000, CST), anti-p65 (1:1000, CST), anti- Histone 3 (1:1000, Abcam), anti-E-cadherin (1:1000, CST), anti-N-cadherin (1:1000, CST) and anti-β-Actin (1:1000, Santa Cruz, USA). The signals were detected by enhanced chemiluminescence (Pierce, USA).
Immunohistochemistry (IHC) staining
The NSCLC and normal lung tissues were fixed, embedded, sectioned, and deparaffinized. Some of the deparaffinized sections were stained with H&E staining. IHC staining was performed using a Dako Envision System (Dako, USA) following the manufacturer’s protocol. Sections were blocked using serum-free protein block buffer (DAKO, CA, USA) for 30 min, after which they were incubated with anti-Id-1 (1:200, Santa Cruz, USA).
Stable cell lines construction
Full-length human Id-1 cDNA was compounded by Genechem and ligated into pENTR221 vector (Genechem, Shanghai). An empty vector was used as the negative control. The Id-1 knockdown was achieved by RNA interference using a lentiviral vector-based shRNA (Genechem, Shanghai). Lentiviral particles corresponding to the shRNA Id-1 NM_002165 target set were used, as well as a nontarget shRNA control. The shRNA sequences were as follows: Id-1 shRNA, CGTGCTCTGTGGGTCTC. For the generation of stable cell lines, Lv-Id-1 or sh-Id-1 was transfected into the NSCLC cells, and selected with puromycin. Specificity and efficacy of the lentivirus were evaluated by Western blotting after transduction and puromycin selection in cells.
Cell proliferation assays
To examine cell proliferation, 5 × 103 cells/well were seeded in a 96-well plate and cultured in a 5% CO2 incubator at 37 °C. At the indicated time points, viable cells were counted using the Cell Counting Kit-8 according the manufacturer’s protocol.
Colony formation assay
Cells were seeded in 6-well plates at a density of 1000 cells per well. After incubation for 14 days, the supernatant was discarded and cells were washed twice in phosphate-buffered saline (PBS). Then, cells were fixed in 10% methanol for 15 min and stained with Giemsa for 20 min. Colonies consisting of more than 50 cells were scored, and the rate of colony formation was calculated.
Cell migration and invasion assay
The migration ability of NSCLC cells was tested in a Transwell Boyden Chamber (8-mm pore size, BD Biosciences) as previously described . For the cell invasion assay, the polycarbonate membranes of the upper compartment of the chambers were precoated with a matrix gel.
The Cells (5× 103) were implanted onto a slide for 24 h, and then were fixed with paraformaldehyde for 30 min, followed permeabilizing with 0.3% Triton X-100/PBS for 5 min at room temperature. Then, cells were blocked with 5% BSA for 1 h at room temperature and stained with anti-E-cadherin (1:200, CST) and anti-N-cadherin (1:200, CST) at 4 °C overnight, followed by incubation with fluorescent-dye conjugated secondary antibody (1:200, Invitrogen) for 1 h, and then stained with DAPI. Fluorescence images were photographed with a confocal microscope.
All data are expressed in terms of means ± the SEM. Significant differences were analyzed using Student’s t-test and two-tailed distribution. The Kaplan-Meier method was used to calculate the survival curve, and log-rank test to determine statistical significance. P values < 0.05 were considered statistically significant.
Id-1 is upregulated in tumor tissues and closely correlated with clinical outcomes of patients with NSCLC
The relationship between Id-1 expression and clinicopathological factors in 96 NSCLC patients
Lymph node metastasis
Id-1 promotes cell viability, migration and invasion of NSCLC cells
Next, we examined the effects of Id-1 on the NSCLC cell’s viability and metastasis. Our results show that H226/sh-Id-1 cells showed a significantly lower in vitro proliferation rate than control cells (Fig. 2b). Conversely, the overexpression of Id-1 significantly increased cell viability of A549 cell (Fig. 2c). Meanwhile, knockdown of Id-1 resulted in a substantial decrease in H226 cell growth in comparison with control groups, whereas overexpression of Id-1 promotes the proliferation of A549 cells by colony formation assay (Fig. 2d and e).
In addition, we investigated the effect of Id-1 on migration and invasion of NSCLC cells. The results demonstrated that knockdown of NSCLC significantly suppressed the cell migration and invasion capability of H226 cells (Fig. 2f), whereas overexpression of Id-1 promoted migration and invasion ability of A549 cells (Fig. 2g). It is well known that EMT is the remarkable presentation for cell metastasis, whether Id-1 knockdown reverse the EMT phenotype need to be identified. As showed in Fig. 2h, western blot revealed that N-cadherin was downregulated after Id-1 knockdown, while E-cadherin was increased in H226 cells. Immunofluorescence assays also confirmed that down-regulation of Id-1 increased the epithelial marker but decreased the mesenchymal maker in H226 cells (Fig. 2i). Therefore, these results demonstrated that down-regulation of Id-1 inhibited NSCLC cell migration and invasion by suppressing the EMT.
Id-1 enhances IκBα phosphorylation and NF-κB activation in NSCLC cells
Next, we detected the effect of Id-1 on the activation of NF-κB in NSCLC cells. As shown in Fig. 3d, overexpression of Id-1 significantly improved the nuclear translocation of p65. Moreover, TNF-α (10 ng/ml) was used to induce the NF-κB activation in Id-1-knockdown cells. The results showed that knockdown of Id-1 led to significant cytoplasm retention of p65 after TNF-α treatment compared with control groups (Fig. 3e and f). Therefore, these findings suggest that Id-1 promoted phosphorylation of IκBα and p65 nuclear translocation in NSCLC cells.
Id-1 promotes migration and invasion of NSCLC cells through NF-κB signaling pathway
Id-1 has been reported to be upregulated and identified as a potential tumor promoter in diverse cancers, including hepatocellular carcinoma and breast cancer [6, 31]. However, it is worth noting that the underlying biological role of Id-1 in NSCLC is still unknown. Here, our findings showed that Id-1 expression remarkable increase in NSCLC tissues compared to the corresponding non-tumor tissues. Importantly, we revealed that high expression level of Id-1 is closely associated with tumor size, high stage, lymph node metastasis, and as an independent risk factor to predict overall survival. These data indicated that high Id-1 expression may represent a novel indicator of poor prognosis in NSCLC and may function as an oncogene in NSCLC progression.
Increasing evidence has demonstrated that Id-1 is involved in the regulation of cancer cell proliferation. Hao et al. have reported that Id-1 promotes osteosarcoma cell growth and inhibits cell apoptosis via PI3K/AKT signaling pathway . Additionally, Id-1 induces ESCC cell proliferation may be through inactivation of p16INK4A/RB pathway . In present study, our experimental data revealed that overexpression of Id-1 significantly enhanced cell proliferation by activation of NF-κB signaling pathway in NSCLC cells. Based on these results, we suggest that the downstream signaling pathways of Id-1-mediated cell proliferation may differ between cancer types.
It is known that metastasis is one of the major characteristics in many cancers, and is associated with cancer progression [27, 30]. Previous studies have demonstrated that Id-1 is suggested to play a critical role during invasion and metastasis in various cancers [11, 18]. For example, Id-1 silencing significantly suppressed the gastric cancer cell migration and invasion in vitro . Cao Y. et al. have reported that fucoidan-induced Id-1 suppression inhibited the in vitro and in vivo invasion of hepatocellular carcinoma cells . Consistent with these studies, our experimental data revealed that downregulation of Id-1 also inhibited migratory ability of NSCLC cells. Thus, these findings suggest that Id-1 would be a potential anti-metastasis therapeutic target for NSCLC patients.
Since Id-1 is a regulator of transcription, it may be responsible for regulation of gene expression involving multiple transduction pathways. So far, several signaling pathways have been suggested to mediate the tumorigenic effects of Id-1 [15, 23]. Id-1 has been shown to promote cell survival by activating NF-κB signaling pathway and it has been suggested that it may be one of the upstream regulators of NF-κB in various cancer [16, 17]. Consistent with this findings, we also found that the NF-κB signaling pathway is responsible for Id-1-mediated tumor cell migration and invasion. As a transcription factors, NF-κB regulates many downstream genes expression including that regulate cell metastasis and plays pivotal roles in both promoting and maintaining an invasive phenotype [3, 20]. Especially, a lots of evidence showed that NF-κB signaling pathway activates the expression of genes important for the progression of NSCLC . For instance, Foxp3 downregulation in NSCLC mediates epithelial-mesenchymal transition via NF-κB signaling pathway . The major finding of this study is that the oncogenic effect of Id-1 is through activation of NF-κB signaling pathway leading to promotion of metastasis in NSCLC cells. On the basis of these findings, Id-1 could be a promising new therapeutic target for suppression the metastasis of NSCLC. Additionally, a previous study agreed with these conclusions revealing that Id-1 is able to protect anticancer drug induced apoptosis through activation of NF-κB pathways in prostate cancer . The NF-κB inhibitor JSH-23 has been evaluated for antitumor activity in a variety of preclinical cancer models [1, 19], but has not been extended to NSCLC. Here, we found that blocking NF-κB signaling pathway using JSH-23 attenuated the Id-1-induced tumor cell metastasis, provide novel evidence to support further explorations into the use of NF-κB inhibitors in NSCLC cancer therapy. Taken together, the tumor-promoting effects of Id-1 in NSCLC cells is at least partly through activation of NF-κB signaling pathway.
In summary, our data revealed that Id-1 was upregulated in NSCLC tissues and cell lines. We also exhibited that Id-1 may be a potential inducement in NSCLC invasiveness though activation of NF-κB signaling pathway. On the basis of these findings, Id-1 could be a promising new therapeutic target for suppression the metastasis of NSCLC.
Availability of data and materials
J. L. and Y. L. conceived and designed the study. J. L., B.W., Y. M., and P. C. performed the experiments. J. L. wrote the paper. J. L. and Y. L. reviewed and edited the manuscript. All authors read and approved the final manuscript.
Ethics approval and consent to participate
Informed consent was obtained from the patients, and the research procedure was approved by the Ethics Committee of The General Hospital of The People’s Liberation Army.
Consent for publication
All authors have read and agreed to the final version of the manuscript.
The authors declare that they have no competing interests.
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