Kruppel-like factor 4 inhibits non–small cell lung cancer cell growth and aggressiveness by stimulating transforming growth factor-β1-meidated ERK/JNK/NF-κB signaling pathways

Abstract

Non–small cell lung cancer is one of the most common epithelial tumors that cause the most common cancer-related mortality due to invasive ability. Research has found that Kruppel-like factor 4, a zinc-finger transcription factor, plays a critical role in the tumor evolution and progression. However, the molecular signal pathways mediated by Kruppel-like factor 4 in the progression of non–small cell lung cancer cells have not been well understood yet. In this study, we investigated the possible role and potential mechanism of Kruppel-like factor 4 in growth and aggressiveness of non–small cell lung cancer cells. Results showed that Kruppel-like factor 4 is downregulated in non–small cell lung cancer cells. Here, we found that Kruppel-like factor 4 knockdown promoted growth and aggressiveness of non–small cell lung cancer cells, as well as enhanced apoptotic resistance induced by tunicamycin. We also found that Kruppel-like factor 4 overexpression significantly suppressed growth and aggressiveness of non–small cell lung cancer cells. Apoptosis rate of non–small cell lung cancer cells induced by tunicamycin was promoted by Kruppel-like factor 4 overexpression. Kruppel-like factor 4 overexpression inhibited transforming growth factor-β1, extracellular signal–regulated protein kinase, C-jun N-terminal kinase, and nuclear factor-κB expression levels in non–small cell lung cancer cells. Mechanistically, Kruppel-like factor 4–mediated tumorigenesis involved suppression of a transforming growth factor-β1-meidated extracellular signal–regulated protein kinase/C-jun N-terminal kinase/nuclear factor-κB transcriptional program in non–small cell lung cancer cells. Our results revealed that Kruppel-like factor 4 overexpression non–small cell lung cancer cell reduces tumor growth in experimental mice. Overall, these data indicate the inhibitory role of Kruppel-like factor 4 in non–small cell lung cancer cells and elaborate a potential molecular signal pathway involving in growth and aggressiveness. Findings identify Kruppel-like factor 4 can be regarded as a possible new molecular agent for designing novel therapeutic protein drug for lung cancer treatment to control non–small cell lung cancer growth.

Keywords

Non–small cell lung cancer Kruppel-like factor 4 aggressiveness apoptosis transforming growth factor-β1 extracellular signal–regulated protein kinase/C-jun N-terminal kinase/nuclear factor-κB

Introduction

Non-small cell lung cancer (NSCLC) is one of the most common human tumors and famous for rapid growth, easy migration, invasion and reoccurrence.¹ A retrospective review has revealed that hypoxia-induced NSCLC cell invasion and migration play a critical role in the tumor progression.² Reports also indicate that NSCLC is a highly aggressive cancer through delocalization of the focal adhesion complex, which presents poor prognosis worldwide.^3,4 Despite numerous therapeutic strategies for the treatment of NSCLC have been clarified, the 5-year survival rate of NSCLC patients was poor.^5–7 Therefore, understanding potential mechanism of local migration and distance invasion of NSCLC is crucial for therapeutic schedule for cancer patients.^8–10

Kruppel-like factor 4 (KLF4) is a zinc-finger transcription factor with diverse functions in various cancer types that is associated with tumorigenesis and progression.¹¹ Although reports have indicated that KLF4 is overexpressed in most human cancer cells, the potential mechanisms of KLF4-regulated cellular metabolism and processes have not been comprehensively understood.^12,13 Evidences have suggested that KLF4 can regulate tumor cells growth, proliferation, differentiation, and apoptotic resistance.^12,14 Zhou et al.¹⁵ have indicated that downregulation of KLF4 expression suppresses lung cancer cell invasion by inhibition of SPARC (secreted protein, acidic and rich in cysteine) expression. Also, characteristic of KLF4 expression with the clinical characteristics of NSCLC tissues was also investigated.¹⁶ These reports indicate that KLF4 expression is involved in the initiation and progression of NSCLC.

Transforming growth factor-β1 (TGF-β1) is a multifunctional cytokine with regulatory effects on tumor cell metabolic processes, which is correlated with the biological parameters into the dosimetric data.¹⁷ Inhibitors on extracellular signal–regulated protein kinase (ERK) phosphorylation could enhance apoptosis and decrease cell viability in NSCLC cells mediated by inhibition of the Ras/Raf/MEK signaling cascade.¹⁸ In addition, downregulation of JNK signaling promotes transfer radiation–induced apoptosis in NSCLC cells.¹⁹ Furthermore, inhibition of nuclear factor-κB (NF-κB) expression and activity contributes to inhibition of lung cancer cell growth and promotes anti-cancer drug-induced apoptosis of cancer cells.^20,21 These reports suggest that TGF-β1, ERK, C-jun N-terminal kinase (JNK), and NF-κB are involved in the progression of NSCLC.

In this study, we investigated potential signaling pathway mediated by KLF4 in NSCLC cells. Our results showed that KLF4 is aberrantly downregulated in NSCLC cells, and knockdown of KLF4 promotes growth and aggressiveness of NSCLC cells through suppression of ERK/JNK/NF-κB signaling pathways. Findings indicate that inhibition of TGF-β1-mediated ERK/JNK/NF-κB activity contributes to the anti-cancer drug cytotoxicity in NSCLC cells, which suggest that KLF4 could be regarded as anti-cancer agent in the treatment of NSCLC.

Materials and methods

Ethics statement

This study was approved by the Protection of Human Subjects Committee of the China. The experimental protocols and animal care were performed and approved by the Animal Care and Use Committee of the China. All surgeries were performed under sodium pentobarbital anesthesia (50 mg/kg) and efforts were made to minimize suffering for experimental mice.

Cells culture

A549 tumor cell lines and MRC-5 normal lung cell line were purchased from American Type Culture Collection (ATCC). A549 cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 100 U/mL penicillin/streptomycin (Sigma-Aldrich, St Louis, MO, USA). MRC-5 cells were cultured in RPMI1640 supplemented with 10% FBS and 100 U/mL penicillin/streptomycin (Sigma-Aldrich). All cells were cultured overnight in serum-free medium before subjecting to the experiments.

Cell growth analysis

A549 was used to assess the effects of KLF4 on NSCLC cells growth. A total of 1 × 10³ cells were seeded into 96-well plates in 100 µL of 10% FBS/DMEM medium. Cells were incubated for 48 h at 37°C in 5% CO₂. After incubation, each well was treated with 20 µL of Cell Titer 96 Aqueous One Solution (Promega) and continued to culture for 1 h. The OD was measured by a microplate reader (Molecular Devices, Sunnyvale, CA, USA) at wavelength of 490 nm.

In vitro migration and invasion assay

Migration and invasion assays were performed in cells’ aggressiveness assay as described in previous report.²² A total of 1 × 10⁵ A549 cells with 500 µL serum-free DMEM were subjected to the tops of BD BioCoat Matrigel Migration or Invasion Chambers (BD Biosciences) according to the manufacturer’s instructions.²³ The number of migrated or invaded A549 cells was determined by counting the cells number in the field at 100× magnification. The tumor cells’ migration and invasion were counted in at least three randomly stain-field microscope every membrane.

Western blot analysis

Cells were homogenized in lysate buffer containing protease-inhibitor and were centrifuged at 8000 r/min at 4°C for 10 min and Western blot assay was conducted using method described previously.²⁴ The rabbit anti-human antibody against KLF4 (1:500, ab72543; Abcam), ERK (1:500, ab54230; Abcam), JNK (1:500, ab179461; Abcam), p65 (1:500, ab16502; Abcam), TGF-β1 (1:500, ab92486; Abcam), tumor necrosis factor (TNF) receptor–associated factor 2 (TRAF2) (1:500, ab126758; Abcam), NF-κB (1:500, ab7204; Abcam), Caspase-9 (1:500, ab52298; Abcam), Caspase-3 (1:500, ab13847; Abcam), Bcl-2 (1:500, ab692; Abcam), Bcl-xL (1:500, ab178844; Abcam), Fas-associated protein with death domain (FADD) (1:500, ab24533; Abcam), TNF-α (1:500, ab6671; Abcam), phospho-ERK (1:400,Thr202/Tyr204, ab201015; Abcam), phospho-JNK (Thr183/Tyr185, 1:400, ab124956; Abcam) and phospho-NF-κB (Ser529, 1:400, ab47395; Abcam), and β-actin (1:500, ab8226; Abcam) were incubated for 2 h at 37°C. All samples were washed with Tris-buffered saline and incubated with horseradish peroxidase (HRP)-conjugated anti-rabbit IgG antibodies (1:2000, Santa Cruz). The results were visualized by using chemiluminescence detection system (BD Biosciences).

Apoptosis assay

A549 cells were cultured until 90% confluence was reached. Apoptosis was assessed by incubating these cells with tunicamycin for 48 h at 37°C in 5% CO₂. After incubation with the tunicamycin, as described above, the cells were trypsinized and collected. The cells were then washed in cold phosphate-buffered saline (PBS), adjusted to 1 × 10⁶ cells/mL with PBS, labeled with Annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) (Annexin V-FITC Kit; BD, San Diego, CA, USA). Numbers of apoptotic cells were analyzed with a FACScan flow cytometer (BD, San Jose, CA, USA).²⁵

Construction of Lentivirus for KLF4 or TGF-β1 overexpression

The KLF4 or TGF-β1 was constructed into Lentivirus plasmid using Lentivirus vector system (System Biosciences, Inc.) with vector as control and named pVector, pKLF4, and pTGF-β1, respectively. All DNA sequences were synthesized by Invitrogen. All of the plasmids were confirmed by DNA sequencing. Plasmids of pVector, pKLF4, or pTGF-β1 were transfected into A549 cells using lipofectamine 2000 according to previously reported studies.²⁶ The cells transfected with pVector, pKLF4, or pTGF-β1 were used for further analysis.

Transfection of small interference RNA

Small interference RNA (siRNA) was used to further determine the potential mechanism of KLF4 in the progress of A549 cells. All siRNAs were synthesized by Invitrogen (Shanghai, China) including siRNA-KLF4 (Si-KLF4) or siRNA-vector (Si-vector). The siRNAs were transfected into A549 cells using HiPer-Fect Transfection Reagent (Qiagen, Valencia, CA).²⁷ The protein expression of each knockdown gene was confirmed by Western blot.

Terminal deoxynucleotidyl transferase–mediated nick-end labeling assay

Terminal deoxynucleotidyl transferase–mediated nick-end labeling (TUNEL; Biotool) was used for analyzing nuclei of apoptotic cells. KLF4-overexpressed or KLF4-knockdowned tumor tissues were incubated with TUNEL for 2 h and then settled on glass coverslips. Subsequently, in situ cell death detection kit was used to observe Fluorescein (Roche) according to the manufacturer’s protocol. Finally, tumor cell images were captured with a ZEISS LSM 510 confocal microscope at 488 nm.²⁸

Animal study

Female BALB/c nude mice (6–8 weeks old; body weight: 30-40 g; n = 40) were purchased from Charles River Laboratories (Berlin, Germany). Non-treated A549, KLF4-overxpressed or KLF4-knockdown A549 cells (1 × 10⁷) were injected into the subcutis of the armpit of experimental mice.²⁹ The tumor volume (V) was determined according to the following equation: V = ab²/2, where a and b are the length and the width, respectively.³⁰ The experimental animals were observed in 21 days.

Immunohistochemistry

Paraffin-embedded tissue sections were prepared and deparaffinized and incubated in 3% hydrogen peroxide for 20 min at 37°C. Tumor sections were blocked by a regular blocking solution (10% goat serum and 1% bovine serum albumin (BSA)) for 20 min at 37°C. All samples were incubated with TGF-β1, NF-κB, ERK, JNK (1:500 dilution; Santa Cruz), phospho-ERK (1:500, Thr202/Tyr204, 9101; Cell Signaling Technology, Inc.), and phospho-JNK (1:500, Thr183/Tyr185, 9255; Cell Signaling Technology, Inc.), respectively, at 4°C for 12 h. Subsequently, tumor sections were washed three times with PBS and incubated with HRP-conjugated anti-rabbit Fab antibodies (1:200, Jackson) for 2 h at 37°C. Quantitative analysis of immunohistochemistry protein expression levels in tumor sections was determined by the means of six-random views in the microscope.³¹

Statistical analysis

The data are presented as mean ± standard deviation (SD) of three independent experiments. Statistical significance was calculated by Student’s t-test using GraphPad Prism 6.0 software (La Jolla, CA, USA). *p < 0.05 and **p < 0.01 were considered as statistical significance.

Results

KLF4 inhibits A549 cells growth, expression levels of TGF-β1, and activation of NF-κB

We initially investigated the KLF4 expression in 20 paraffin-embedded human NSCLC specimens and their corresponding adjacent normal lung tissues. As shown in Figure 1(a), KLF4 expression levels were downregulated in NSCLC specimens compared to normal lung tissues (p < 0.01). Western blot demonstrated KLF4 protein levels also decreased in NSCLC specimens and tumors compared to normal tissue and MRC-5 cells (p < 0.01; Figure 1(b)). We also demonstrated that KLF4 administration inhibited A549 cells growth, while knockdown of KLF4 promoted A549 cells growth (p < 0.01; Figure 1(c)). KLF4 inhibited expression levels of TGF-β1 and stimulated NF-κB activation in A549 cells, while KLF4 knockdown promoted TGF-β1 expression and suppressed NF-κB activation in A549 cells (p < 0.01; Figure 1(d) and (e)). These results suggest that KLF4 expression is downregulated in NSCLC and could inhibit A549 cells growth, expression levels of TGF-β1, and activation of NF-κB.

Figure 1.

KLF4 inhibits A549 cells growth TGF-β1, expression and NF-κB activity. (a) Comparison of KLF4 expression between NSCLC tissues and adjacent normal lung tissues determined by immunohistochemistry. (b) Protein levels of KLF4 in NSCLC and adjacent normal lung tissues and cells. (c) Effects of overexpression or knockdown of KLF4 on A549 cells growth. (d) Effects of overexpression or knockdown of KLF4 on TGF-β1 expression. (e) Effects of overexpression or knockdown of KLF4 on NF-κB activation.

KLF4 suppresses A549 cells aggressiveness and ERK and JNK expression and phosphorylation

Effects of KLF4 on aggressiveness and ERK and JNK expression and phosphorylation levels were analyzed in A549 cells. We found that KLF4 blocked migration and invasion of A549 cells and knockdown of KLF4 promoted aggressiveness of A549 cells (p < 0.01; Figure 2(a)–(d)). Western blot demonstrated that KLF4 suppresses ERK and JNK expression and phosphorylation, while KLF4 knockdown promoted ERK and JNK expression and phosphorylation in A549 cells (p < 0.01; Figure 2(e) and (f)). These results indicate that KLF4 suppresses A549 cells aggressiveness and downregulates ERK and JNK expression and phosphorylation in A549 cells.

Figure 2.

KLF4 suppresses A549 cells aggressiveness and ERK and JNK expression and phosphorylation. (a) KLF4 suppresses A549 cells migration. (b) Knockdown of KLF4 promotes A549 cells migration. (c) KLF4 suppresses A549 cells invasion. (d) Knockdown of KLF4 promotes A549 cells invasion. (e). KLF4 suppresses ERK and JNK expression and phosphorylation in A549 cells. (f) Knockdown of KLF4 promotes ERK and JNK expression and phosphorylation in A549 cells.

KLF4 enhances apoptosis of A549 cells induced by tunicamycin

Next, we analyzed the role of KLF4 on apoptotic resistance of A549 cells induced by tunicamycin in vitro. Results showed that cleaved Caspase-9 and Caspase-3 were upregulated by KLF4, while were downregulated by KLF4 knockdown in A549 cells (p < 0.01; Figure 3(a) and (b)). Anti-apoptosis gene Bcl-2 and Bcl-xL expression levels were significantly downregulated by KLF4 and upregulated by KLF4 knockdown in A549 cells (p < 0.01; Figure 3(c) and (d)). We observed KLF4 knockdown inhibited and KLF4 promoted apoptosis of A549 cells induced by tunicamycin (p < 0.01; Figure 3(e) and (f)). Notably, we found that FADD and TNF-α expression levels were also increased by KLF4 and decreased by KLF4 knockdown (p < 0.01, Figure 3(g) and (h)). These results indicate KLF4 enhances apoptosis of A549 cells induced by tunicamycin.

Figure 3.

KLF4 promotes apoptosis of A549 cells induced by tunicamycin. (a) KLF4 promotes and (b) KLF4 knockdown inhibits expression levels of Caspase-9 and Caspase-3 in A549 cells. (c) KLF4 inhibits and (d) KLF4 knockdown promotes gene Bcl-2 and Bcl-xL expression levels in A549 cells. (e) KLF4 promotes and (f) KLF4 knockdown inhibits apoptosis of A549 cells. (g) KLF4 enhances and (h) KLF4 knockdown inhibits FADD and TNF-α expression levels in A549 cells.

KLF4 regulates A549 cells growth via TGF-β1-mediated ERK/JNK/NF-κB signaling pathway

To analyze potential mechanism of KLF4-mediated growth and aggressiveness, we investigated TGF-β1-mediated ERK/JNK/NF-κB signaling pathway in A549 cells. As shown in Figure 4(a) and (b), KLF4 increased NF-κB expression levels and NF-κB-regulated gene p65 and TRAF2 compared to control, while its target genes were decreased by KLF4 knockdown (p < 0.01). We found that overexpression of TGF-β1 abrogated KLF4-inhibited (pTGF-β1-K4) expression and phosphorylation levels ERK, JNK, and NF-κB in A549 cells (p > 0.05; Figure 4(c) and (d)). Potential mechanism analyses showed that overexpression of TGF-β1 blocked KLF4-prmoted apoptosis of A549 cells induced by tunicamycin (p > 0.05; Figure 4(e)). Interestingly, overexpression of TGF-β1 also canceled KLF4-inhibited growth, migration, and invasion of A549 cells (p > 0.05; Figure 4(f)–(h)). These results indicate that KLF4 regulates A549 cells growth and aggressiveness via TGF-β1-mediated ERK/JNK/NF-κB signaling pathway.

Figure 4.

KLF4 regulates A549 cells growth through regulation of TGF-β1-mediated ERK/JNK/NF-κB signaling pathway. (a) KLF4 increases NF-κB expression levels and NF-κB-regulated gene p65 and TRAF2 in A549 cells. (b) KLF4 knockdown decreases NF-κB expression levels and NF-κB-regulated gene p65 and TRAF2 in A549 cells. Overexpression of TGF-β1 abrogates KLF4-inhibited (pTGF-β1-K4) (c) expression and (d) phosphorylation levels of ERK, JNK, and NF-κB in A549 cells. (e) Overexpression of TGF-β1 blocks KLF4-promoted apoptosis of A549 cells induced by tunicamycin. Overexpression of TGF-β1 cancels KLF4-inhibited (f) growth, (g) migration, and (h) invasion of A549 cells.

KLF4 inhibits NSCLC tumor growth in vivo

We further investigated in vivo assays to identify the inhibitory effects of KLF4 in DBA/2 mice. Results showed that KLF4-overexpressed significantly inhibited tumor growth, while KLF4-knockdown promoted tumor growth in vivo (Figure 5(a)). Histological staining indicated that KLF4 suppressed TGF-β1 and increased NF-κB expression levels in tumors (Figure 5(b)). Immunohistochemical analyses demonstrated expression and phosphorylation levels of ERK and JNK in tumor tissues (Figure 5(c)). Results also indicated that apoptotic tumor cells were increased in KLF4-treated tumors in vivo (Figure 5(d)). These results suggest that KLF4 can inhibit NSCLC tumor growth in vivo.

Figure 5.

KLF4 inhibits NSCLC tumor growth in vivo. (a) Effects of KLF4 on tumor growth in BALB/c nude mice. (b) KLF4 suppresses TGF-β1 and increased NF-κB expression levels in tumors isolated from experimental mice. (c) Effects of KLF4 on expression and phosphorylation levels of ERK and JNK in tumor tissues. (d) KLF4 increases the number of apoptotic tumor cells in tumor tissues in vivo.

Discussion

Rapid tumor proliferation and stronger invasive ability are the most important characteristics of NSCLC, which have been clearly demonstrated in a large number of reports.^32,33 Currently, KLF4 is reported to present diverse functions that can be acted as a tumor suppressor or an oncogene depending on different cellular contexts.^16,34,35 In this study, we further investigate the inhibitory effects and potential molecular signal pathway mediated by KLF4 in the progression of NSCLC. Our results indicate that KLF4 is downregulated in NSCLC specimens and cells that are involved in growth, apoptotic resistance, and aggressiveness of NSCLC through regulation of TGF-β1-mediated ERK/JNK/NF-κB signaling pathway. Findings suggest that KLF4 can inhibit NSCLC tumor growth both in vitro and in vivo.

KLF4-Numb-like signaling can regulate lung cancer metastasis that is critical to regulate metastatic progression.³⁶ Yu et al.³⁷ have revealed that KLF4 can regulate adult lung tumor–initiating cells and repress K-Ras-mediated lung cancer, which presents the inhibitory effects on proliferation of lung cancer cells. In addition, research suggests that KLF4 plays simulative role in hydrogen-peroxide-induced apoptosis of chronic myeloid leukemia cells via regulation of the Bcl-2/bax pathway.³⁸ Furthermore, targeting of KLF4 contributes to apoptosis induced by Adriamycin in osteosarcoma cells.³⁹ In this study, our results demonstrated that KLF4 treatment inhibits growth and aggressiveness, while knockdown of KLF4 promotes growth and aggressiveness of NSCLC cells. Importantly, we found that KLF4 overexpression decreases apoptotic resistance of A549 cells induced by tunicamycin through regulation of cleaved Caspase-9, Caspase-3, Bcl-2, and Bcl-xL expression levels, which is agreed with previously reported studies.^40,41

KLF4-mediated inhibition of tumor cells is probably repressed by TGF-β1 and that through downregulation of AT1 receptor expression via protein kinase C (PKC)-delta-mediated Sp1 dissociation in vascular smooth muscle cells.⁴² In this study, we investigated the regulatory effects of KLF4 on TGF-β1 expression in NSCLC cells and results indicate that KLF4 inhibits TGF-β1 expression, which suppresses growth and aggressiveness of A549 cells. Morita et al.⁴³ have indicated that BAALC can potentiate oncogenic ERK pathway through interactions with MEKK1 and KLF4. Interestingly, KLF4 is regulated by RAS/RAF/MEK/ERK signaling through E2F1 and promotes melanoma cell growth, and findings have identified that KLF4 may be a possible new molecular target for designing novel therapeutic treatments to inhibit melanoma growth.⁴⁴ Reversely, our findings showed that KLF4 is decreased in NSCLC, and findings have suggested that KLF4 can regulate A549 cells growth through downregulation of TGF-β1-mediated ERK/JNK/NF-κB signaling pathway in NSCLC both in vitro and in vivo. Knockdown of KLF4 expression levels significantly upregulates anti-apoptosis gene expression that further stimulates tumor growth by promoting TGF-β1-mediated ERK/JNK/NF-κB signaling pathway. However, the inhibitory effects of KLF4 on NSCLC growth and progression as well as the in vivo suppressive role of in human NSCLC pathogenesis on the metastasis of NSCLC should be further investigated.

In summary, our results demonstrated that KLF4 can be regarded as a tumor suppressor agent in the progression of NSCLC pathogenesis. Findings indicate that KLF4 not only inhibits growth and aggressiveness but also promotes apoptotic sensibility of NSCLC for anti-cancer drugs, which suggest that KLF4 may be a potential biomarker and are valuable anti-cancer agent for the treatment of NSCLC.

Footnotes

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

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. KLF4 is regulated by RAS/RAF/MEK/ERK signaling through E2F1 and promotes melanoma cell growth. Oncogene. Epub ahead of print 9 January 2017. DOI: 10.1038/onc.2016.481.