Mechanisms of Activation of NFκB Signaling Pathway

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Objective: we aimed to analyze the mechanisms of activation of NFκB signaling pathway in the carcinogenesis of LSCC.

Methods: Protein array was used to identify the differentially expressed proteins involved in NFκB signaling pathway in 50 LSCC cells compared with 50 cases of normal throat mucosa. Correlation analysis between significantly differentially expressed proteins and clinical characteristics was also performed. The expression of IKK-β was detected both by RT-PCR and Western-blot in Hep-2 cells transfected with IKK-β-siRNA or pcDNA3.1-IKK-β. Besides, MTT, flow cytometer, and Transwell assay was respectively used to examine the proliferation, apoptosis, and migration rate of Hep-2 cells.

Results: Three differentially expressed proteins were identified. Among these, TNFR1 and IKK-β were significantly up-regulated (P < 0.01) and FADD1 was significantly down-regulated (P < 0.01). The correlation analysis showed that IKK-β had significant association with histological grade, clinical stage, and node metastasis (P < 0.05). Besides, the high expression of IKK-β resulted in increased proliferation and significant migration rate (P < 0.05) compared with Hep-2 cells. Reversely, Hep-2 cells transfected with IKK-β-siRNA showed significant lower proliferation and migration rate (P < 0.05), and significant higher apoptosis rate (P < 0.05) than normal cells.

Conclusions: High expression of IKK-β can promote the growth and invasion of LSCC cells. Thus, the activation of NFκB signaling pathway through IKK-β plays an important role in the pathogenesis and development of LSCC, and suppression of IKK-β expression may be an efficient therapeutic approach for inhibition of LSCC.

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Keywords: Laryngeal squamous cell carcinoma; progression; NFκB signaling pathway; IKKβ; siRNA

Introduction

Laryngeal squamous cell carcinoma (LSCC) is the second most common malignant tumors of head and neck (1). The incidence rate is high in Southeast Asia and Eastern Europe, and the trend is increasing around the world (2). In LSCC, the various tumor growth rate is an important factor for survival (3). Early stage LSCC is often curable with surgery or radiotherapy. Currently, tumor site, clinical stage, histopathologic grade, molecular and cellular characteristics of the primary tumor is used to evaluate prognostic (4).

Nuclear factor-κB (NFκB), a transcription factor, is also widely investigated in many fields for its complexity subunits, multiple genes that it regulates, unusual and rapid regulation, major role in immunology, and involvement in many diseases (5). NFκB is responsible for DNA binding, dimerization, and interaction with inhibitory factors (IκB proteins). In the mammalian cells, NFκB is activated by pro-inflammatory cytokines and extracellular stimuli through contact with specific receptors including tumor necrosis factor receptor 1 or 2 (FNFR1/2), T-cell receptor (TCR), interleukin-1 (IL-1) receptor, and TLR (6). It is known that NFκB activity speeds cancer cells proliferation, inhibits apoptosis, promotes angiogenesis and induces epithelial-mesenchymal transition. While suppression of NFκB in tumor cells generally leads to tumor regression (7). NFκB is constitutively deregulated in several human cancers, including pancreas, breast, liver and stomach (8-10). It has also been reported that aberrant NFκB activation leads to oncogenes expression and squamous cell carcinoma of head and neck (HNSCC) (11).

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Although the nuclear localization of NFκB and p53 plays an important role in the development of LSCC (12), the activation mechanism of NFκB signaling pathway involved in the carcinogenesis of LSCC remains unclear. In the present study, we used protein array to analyze the differentially expressed proteins related to NFκB signaling pathway in LSCC compared with normal tissues. Effects of the most differentially expressed protein on proliferation, apoptosis, and migration were further analyzed using over-expression or RAN interfering method in order to understand the correlation between NFκB and LSCC.

Materials and methods

Clinicopathologic characteristics of patients

The LSCC cases and normal tissues were recruited from the Department of Otorhinolaryngology at the Second peoples’ hospital in Shanghai. A total of 50 LSCC patients (aged from 40 to 62) confirmed by pathologists were included in this study. The average and medium age was 54 and 51, respectively. The ratio of male: female was 1: 1. Among the 50 LSCC patients, 38 patients had node metastasis. According to differential degree, 24 cases were well-differentiated, 18 medium-differentiated and 8 low-differentiated. Tumors were histologically graded to 4 stages (I: 5 cases, II: 11 cases, III: 28 cases, IV: 6 cases) based on the tumor node metastasis (TNM) classification of the International Union Against Cancer (UICC). In addition, the control throat mucosa tissues were also collected from 50 paitents (aged from 43 to 60, gender ratio = 1: 1) who had received uvulopalatopharyngoplasty. The average and medium age was 52 and 50, respectively.

Protein array

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LSCC and normal mucosa tissues were lysed by radio-immunoprecipitation assay (RIPA) solution (Beyotime, Shanghai), and the total protein concentration in the supernatant was detected using the bicinchoninic acid (BCA) method (Raybiotech, BCA protein assay kit). The procedures were performed strictly as described in the instruction. Control and tumor samples were each divided into two equal portions. After drying at room temperature for 2h, 100 ul blocking solution was added to each column, and the slides were incubated for 1h under shaking. After incubation, blocking solution was discarded. Tissue lysate was diluted to a final concentration of 500 ug/ml and added to the samples, and then incubated at 4℃ overnight. After washed, CY3-labeled antibody mixture was added and incubated at room temperature for 2h, then the samples were washed again and scanned using GenePix 4000B (MolecularDevices, USA).

Construction of eukaryotic expression vector

Total RNA was extracted using Trizol (GIBCOBRL). Oligo-dT, dNTP, DTT, and reverse transcriptase were used in the synthesization of cDNA. The sequence of IKK-β was amplified from 5 ul cDNA template via PCR using special primers. Forward: 5’-CCAAGCTTCCACCATGAGCTGGTCAC-3’. Reverse: 5’-GGGGTACCTGAGGCCTGCTCCAGGC-3’. The resultant PCR fragment was purified with gel extraction kit, treated with HindIII and KpnI and purified again. Then, the restricted IKK-β fragment was inserted into vector pcDNA3.1 at the same digestion sites to yield pcDNA3.1-IKK-β, which was verified by PCR and sequencing.

siRNA preparation

siRNA was designed according to IKK-β mRAN sequence applied in Human Gene database using Ambion software, and synthesized by Borui Biology (Guangzhou). The target sequence was CCAATAATCTTAACAGTGT. The sense strand of siRAN was 5’ CCAAUAAUCUUAACAGUGUdTdT 3’. Anti-sense: 3’ dTdTGGUUAUUAGAAUUGUCACA 5’. A non-targeted siRNA was designed as negative control.

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Transfection of Hep-2 cells

Hep-2 cells were obtained from the Chinese academy of science (Shanghai) and cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum and kept at 37℃ under 5% CO₂. Hep-2 cells were separately transfected with IKK-β siRNA, Non-targeted siRNA, pcDNA3.1-IKK-β, or pcDNA3.1 according to the manufacturer’s instructions (Hiperfect Transfection Reagent, Qiagen, USA). Hep-2 cells (2×10⁵ cells/ml) were plated into independent wells of 6-well (2 ml) and 96-well dish (100 ul). Then, 3 μl hiperfect transfection reagents were added and mixed gently. After incubated for 10min at room temperature, cells mixture was slowly added to the wells at 37℃ under 5% CO₂ for 6h. Subsequently, transfection reagent was replaced by DEME medium and incubated for 24h, 48h, and 72h, respectively.

RT-PCR and Western blot

RT-PCR: Total RNA was extracted from cells incubating separately for 24h, 28, 72h as described above. RNA was quantified and cDNA was synthesized. The primers for IKK-β were 5’-GACTTGAATGGAACGGTGAA-3’ (Forward) and 5’-TCTTGGGCTCTTGAAGGATA-3’ (Reverse). The primers for β-actin were 5’-CTCCCACCTTATCTACTCCC-3’ (Forward) and 5’-TAGCTGCTCGCTGTCTTG-3’ (Reverse). Amplification conditions: 94℃, 5min; 95℃, 30s; 55℃, 30s; 72℃, 30 cycles of 30s; 72℃, 10min. The PCR products were isolated by agarose gel electrophoresis and quantified by a Gel imager. β-actin was used as an internal control.

Western blot: Cells were lysed and total protein concentration was detected. A total of 50 ul lysate was sampled to SDS-PAGE. After electrophoresis, the separated target proteins were transferred to a polyvinylidene flroride (PVDF) membrane and blotted with 5% non-fat dry milk. Then, the membrane was incubated overnight with anti-IKK-β or anti-β-actin (purchased from Abcam). After incubation with horseradish peroxidase-conjugated anti-mouse secondary antibody (IgG, Abcam) and washed, the protein bands were detected with chemiluminescence.

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MTT assay

MTT (3- (4, 5-dimethylthiazole-2-yl)-2, 5-diphenyltetrazolium bromide) assay was used to determine the viability of Hep-2 cells cultured for 24h, 48h, and 72h ,respectively. Cells grown in 96-well multiplates were added with 20 μl MTT (5 mg/mL) and incubated for 4h. Then, the supernant was discarded and 150 μl DMSO was added. After incubation for 10min with shaking, the final product was quantified spectrophotometrically by absorbance at 570 nm wavelength.

Flow cytometry

Cellular apoptosis was assessed by flow cytometry. Trypsinized cells were collected at 2000 rpm × 10min and washed three times with phosphate-buffered saline (PBS). Propidium iodide (PI) was added and the cells were incubated for 15min at 4℃ in dark. The stained cells were analyzed by Elite Esp flow cytometer.

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Transwell migration assay

For cell migration assay, 5 × 10⁵ cells were added to the upper chamber and the lower chamber was filled with 500 μl medium without fetal bovine serum. An 8 μm polycarbonate membrane was placed between the two champers. After migration for 24h at 37℃ under 5% CO₂, cells in the upper chamber of transwell filter were removed with a cotton swab. Then, the membrane was fixed in methanol for 10min, stained with Giemsa for 20min. The migration number was counted in 5, 400 × microscopic fields per filter.

Statistical analysis

Statistical analysis was performed using SPSS12.0 software. Comparisons between two groups were accomplished using t test, and correlation analyses between differentially expressed proteins and clinicopathologic characteristics were conducted by Sperman analysis. P < 0.05 was considered to be statistically significant.

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Results

TNFR1, IKK-β and FADD were differentially expressed in LSCC cells

Protein array was used to detect the differentially expressed cytokines in LSCC cells compared with normal tissues. The result showed that both TNFR1 (tumor necrosis factor receptor 1) and IKK-β (IκB kinase-β) were significantly over-expressed, FADD (Fas (TNFRSF6)-associated via death domain) was significantly down-expressed in LSCC cells (P < 0.01, Fig 1 & Table 1). Furthermore, we analyzed the correlation between the differently expressed cytokines and clinicopathological characteristcs. As shown in Table 2, the expression of IKK-β was significantly high in poorly (medium and low) -differentiated (3482.31, high: 1775.48; P < 0.05), histologic grade III-IV (3452.95, I-II: 1834.42; P < 0.05), or node metastatic tissues (3449.58, without: 1874.25; P < 0.05). The correlations between the other two cytokines and clinicopathological characteristics were not significant (P > 0.05).

Construction of expression plasmid pcDNA3.1-IKK-β

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The recombinant plasmid pcDNA3.1-IKK-β was verified by PCR and sequencing. As shown in Fig 2, a 2270 bp fragment was amplified which was consistent with the length of IKK-β gene, and the sequencing result was also the same as reported in GenBank.

The expression of IKK-β in Hep-2 cells transfected with pcDNA3.1-IKK-β or IKK-β-siRNA

We detected the expression level of IKK-β using semiquantitative RT-PCR and Western blot in Hep-2 cells transfected with pcDNA3.1-IKK-β or IKK-β-siRNA at 24h, 48h, and 72h, respectively. The results were shown in Fig 3. The amplified fragment of IKK-β was about 520 bp. When cells were transfected with IKK-β-siRNA, the transcription of IKK-β mRAN was obviously down-regulated over time (Fig 3A). Reversely, when cells were transfected with pcDNA3.1-IKK-β, IKK-β mRAN transcription was significantly up-regulated with the incubating time lasting (Fig 3B). Western blot showed the same results (Fig 3C, D). The expression of IKK-β was not changeable in normal Hep-2 cells or cells transfected with Non-targeted siRNA.

Proliferation, apoptosis, and migration rate in Hep-2 cells transfected with pcDNA3.1-IKK-β or IKK-β siRNA

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The MTT assay was used to calculate the proliferation rate in transfected Hep-2 cells incubated at 24h, 48h, or 72h. As shown in Fig 4A, the correlation between proliferation rate and IKK-β expression was positively significant. The proliferation rate of Non-targeted siRNA or pcDNA3.1 transfected cells was not been affected compared with control group (P > 0.05). While, the proliferation rate of cells trancfected with IKK-β siRNA was significantly decreased (P < 0.05), and the rate was significantly increased in cells transfected with pcDNA3.1-IKK-β (P < 0.05). Besides, there was a positive association between proliferation rate and incubation time, such as pcDNA3.1-IKK-β transfected cell whose proliferation rate was increased from 150% (24h) to 260% (72h).

Cells apoptosis rate was also analyzed by flow cytometry at 24h, 48h, and 72h, respectively. Results were shown in Fig 4B. The apoptosis rate of cells transfected pcDNA3.1-IKK-β or pcDNA3.1 or Non-targeted siRNA was similar with the control Hep-2 cells, while the apoptosis rate of IKK-β siRNA transfected cells was significantly increased (from 18% at 24h to 60% at 72h) compared with control cells (P < 0.05).

Moreover, cells migration rate was achieved through Transwell analysis. As shown in Fig 4C, the rate was similar in Non-targeted siRNA, pcDNA3.1 transfected cells and control Hep-2 cells. However, it was significantly reduced (from 20% at 24h to 5% at 72h) in IKK-β siRNA transfected cells (P < 0.05), and was significantly increased (from 50% at 24h to 90% at 72h) in pcDNA3.1-IKK-β transfected cells compared with control Hep-2 cells (P < 0.05).

Discussion

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The development and migration of LSCC is a complex process which is associated with changeable expression of multiple genes, including p53, p21, BCL-2 (B-cell CLL/lymphoma 2), CD34, CD44, Ki-67, CylinD1, c-Myc, and FHIT (fragile histidine triad) (13-16). In the present study, three differentially expressed cytokines (TNFR1, IKK-β and FADD) involved in NFκB signaling pathway were identified in LSCC cells compared with control cells using protein array. Among those, IKK-β had significant correlation with clinicopathological characteristics. Furthermore, we found that IKK-β expression had significant effects on proliferation, apoptosis, and migration of Hep-2 cells.

IKK complex consists of IKKα (IKK1), IKKβ (IKK2), and IKKγ. It has been believed that IκB proteins inhibit NFκB activity mainly by masking the nuclear localization signal and DNA binding activity, and thus deregulating transcription of genes controlled by NFκB (17,18). Hence, removing of IκB proteins is necessary to active NFκB. IκB proteins are proteolysed through phosphorylation on special serine residues by IKK proteins. For instance, IKKβ activiation by Toll-like receptor (TLR) is necessary and sufficient for phosphorylation of IκBα at Ser-32 and Ser-36 (19).

NFκB can be initiated by different pathways. The classical pathway is triggered by activation TNF receptor-associated factor (TRAF) adapter proteins and the subsequent stimulation of the IKK complex (9,20). TNFR1, a type transmembrane glycoprotein, is one of the TNF-α homologous receptors. Upon binding of TNF-α, the activation of NFκB and MAPKs (Mitogen-activated protein kinases) (21) are induced. Ligation between TNFR1 and its adapter proteins including TRADD (TNF receptor-associated protein with a death domain) and RIP1 (Serine–threonine kinase receptor-interacting protein 1) is carried out through a death domain (22). When associated with TNFR1, TRADD further recruits TRAF2, which forms complexity with cellular inhibitor of apoptosis protein (cIAP) -1and cIAP-2 via its N-terminal RING domain (23,24). Thus, a signaling core complex containing TNFR1, RIP1, TRAF2, cIAP-12IKK proteins is formed, and this complex recruit TGF-β activated kinase (TAK) 1 and IKK proteins (25).

On the other hand, FADD is a common conduit in TNF-α mediated apoptosis where a death inducing signaling complex comprised of TRADD, FADD and pro-caspase-8 is formed (26). This complex activates pro-caspase-8 and a cascade downstream apoptotic signaling including BID, Cytochrome c, Apaf-1 , caspase-9, and caspase-3 are initiated (27,28). It has been reported that FADD level is increased in cells exposed to TNF or senditizing agent, and its over-expression can initiate the apoptotic death signaling pathway even without a death ligand (29,30). In our study, TNFR1 and IKK-β was high-expressed and FADD was down-expressed in LSCC cells compared with control. These results suggest that in LSCC tumor cells both apoptotic pathway involved FADD and anti-apoptotic pathway contained IKK-β is initiated. However, the anti-apoptotic pathway is dominant. NF-κB signaling pathway is activated by the integration of many participants caused by association of over-expressed TNFR1 and TNF-alpha thereby induces activation of IKK complex to phosphorylate IκB.

In addition, we found that the expression level of IKK-β is significantly correlated with poorly-differentiation, high histologic grade and node metastasis in our study. Therefore, we analyzed the effects of interfering or over-expression of IKK-β on cells proliferation, apoptosis, and migration. Tang et al. reported that activation of genes along NFκB pathway including NIK, IKK1, IKK2, and NFκB gene were important in initiation cell proliferation (31).IKK-β-deficient embryonic fibroblasts in micedied at ~14.5 days of gestation because of liver degeneration and apoptosis (32). Additionally, some of the chemokines emerged in the process of NFκB activation can stimulate the migration and maturation of lymphocytes (33). Similarly, in the represent study, over-expression of IKK-β lead to significantly increased proliferation and migration rate, while interfering of IKK-β resulted in significantly decreased proliferation, migration rate and increased apoptosis. It has also been shown that curcumin (an anti-inflammatory agent) suppresses cellular transformation, proliferation, angiogenesis, invasion, and metastasis through inhibition of IKK and AKT activation thereby suppressed NFκB activation (34). These results suggest that IKK-β expression and activity is also essential in LSCC cell proliferation, apoptosis, and invasion regulated by NFκB signaling pathway. Thus, inhibition of IKK-β activity may be served as an effective therapeutic approach for inhibiting carcinogenesis of LSCC cell via inhibiting the NFκB signaling pathway.

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Overall, the activation of NFκB signaling pathway related to TNFR1, IKK-β, and FADD plays a major role in the development of LSCC. Abnormally expressed IKK-β has effects on cells proliferation, apoptosis, and migration. However, this is a multiplex process since there are several chains combining TNFR1 with stimulation of IKK complex and activation of the classical NFκB signaling pathway. Therefore, further knockout or point mutation experiments for one gene or multiple genes involved in NFκB signaling pathway are still needed to study the details in the carcinogenesis of LSCC.