Abstract
Low expression of the tumor suppressor Kelch-like ECH-associated protein 1 (KEAP1) in non–small cell lung cancer (NSCLC) often results in higher malignant biological behavior and poor prognosis; however, the underlying mechanism remains unclear. The present study demonstrates that overexpression of Keap1 significantly suppresses migration and invasion of three different lung cancer cells (A549, H460, and H1299). Highly expressed Keap1, compared with the control, promotes formation of multiple stress fibers with larger mature focal adhesion complexes in the cytoplasm where only fine focal adhesions were observed in the membrane under control conditions. RhoA activity significantly increased when Keap1 was overexpressed, whereas Myosin 9b expression was reduced but could be rescued by proteasome inhibition. Noticeably, mouse tumor xenografts with Keap1 overexpression were smaller in size and less metastatic relative to the control group. Taken together, these results demonstrate that Keap1 stabilizes F-actin cytoskeleton structures and inhibits focal adhesion turnover, thereby restraining the migration and invasion of NSCLC. Therefore, increasing Keap1 or targeting its downstream molecules might provide potential therapeutic benefits for the treatment of patients with NSCLC.
Implications: This study provides mechanistic insight on the metastatic process in NSCLC and suggests that Keap1 and its downstream molecules may be valuable drug targets for NSCLC patients. Mol Cancer Res; 16(3); 508–16. ©2018 AACR.
Introduction
Lung cancer is the leading cause of cancer mortality worldwide, due to its steadily increasing incidence, high mortality, and lack of advanced treatment (1, 2). Non–small cell lung cancer (NSCLC) accounts for approximately 85% of lung cancers (3, 4) and is difficult to detect in the early stages; thus, most patients are diagnosed in the advanced or metastatic cancer stage. The high mortality rate of NSCLC is largely related to its metastatic behavior (5, 6); therefore, understanding this mechanism is crucial for developing effective treatments for NSCLC in the clinic.
Tumor cell motility is important for its invasion and migration. Cell movement is a sequential series of steps initiated by establishing actin-rich protrusions and stabilizing the leading edge by nascent focal adhesions, followed by F-actin contraction, disassembling focal adhesion at the cell rear, and detaching the tail of the cell, all of which cause forward movement of the cell (7, 8). Previous studies have shown that F-actin reorganization and focal adhesion turnover play key roles in tumor cell invasion and migration (9). RhoA, a member of the Rho-GTPase family, is important for the regulation of actin cytoskeleton dynamics and is involved in many cell biological functions including malignant transformation, cell polarity, and cell migration (10–12). RhoA directly promotes stress fiber formation and focal adhesion maturation, which stabilize the cytoskeleton (13–15). Therefore, alterations of RhoA activity may affect cell motility.
In recent years, a large amount of evidence has suggested that Kelch-like ECH-associated protein 1 (Keap1) often functions as a cancer suppressor (16), as its expression was downregulated in different types of solid cancers including NSCLC (17–19). It was well known that Keap1 proteins have five functional domains: N-terminal (NTR), Bric-a-Brac (BTB) dimerization, intervening or linker region (IVR), double glycine repeat (DGR), and C-terminal region (CTR), of which BTB and DGR are characteristic domains (20). The BTB domain is not only involved in Keap1 homodimerization but also contains the binding site for the cul3-dependent E3 ubiquitin ligase complex (21). Thus, Keap1 may also function as a substrate adaptor protein for the E3 ubiquitin ligase complex to promote protein-ubiquitination degradation through the 26S proteasome (22). The DGR domain is essential for binding to actin proteins, which may play a role for stabilizing the cytoskeleton (23). This is supported by the fact that the majority of Keap1 is often colocalized with F-actin cytoskeleton in the cytoplasm. Based on the molecular and structural characteristics, it is believed that Keap1 may promote F-actin formation and inhibit focal adhesion turnover by enhancing RhoA activity. Furthermore, Keap1 can also stabilize F-actin structures by directly binding to actin proteins. Therefore, the aim of this study is to determine the underlying mechanism of Keap1 inhibition for NSCLC cell invasion and migration, with particular focus on the molecular mechanism of Keap1-mediated actin stabilization and upregulation of RhoA activity. Our results suggest that Keap1 and its downstream molecules may be a valuable drug target for patients with NSCLC.
Materials and Methods
Cell culture
Human NSCLC cell lines NCI-H1299, NCI-H460, and A549 were purchased from ATCC and were cultured for no more than 15 passages. NCI-H1299 and NCI-H460 were cultured in RPMI 1640 (Gibco). While A549 cells were cultured in high-glucose Dulbecco's Modified Eagle's Medium (DMEM). Both mediums were supplemented with 10% fetal bovine serum (FBS, Invitrogen) and 1% penicillin/streptomycin solution (P/S, Gibco). Cell lines were authenticated by short tandem repeat (STR) profiling in 2014 (Shanghai Biowing Applied Biotechnology Co. Ltd). All cell lines used were tested negative for mycoplasma contamination by the PCR mycoplasma detection kit (ABM) in 2015.
Lentiviral-based plasmids transduction
The full-length Keap1 gene was cloned and introduced into the Lentivirus vector (Lentivirus carrying Keap1 (LV-NM_203500) or negative control (LV-CON063) GeneChem). Cells in the logarithmic growth phase were seeded in 3.5 cm dish at a density of 5 × 104 cells/well and infected with LV-NM_203500 or LV-CON063 in 5 μg/mL polybrene when the cells reached 30% confluence. Then cells were selected and evaluated for subsequent experiments.
Real-time monitoring of the cell migration and invasion
Dynamic and real assessments of migration and invasion were performed by measuring the cell index, which is proportional to the number of cells, using the xCELLigence System Real-Time Cell Analysis System (RTCA; ACEA Biosciences; refs. 24, 25). Briefly, the RTCA functions on electronic impedance reading from the gold-plated sensor electrodes that are placed at the bottom of the plats (E-16 plate). Electronic reading change as cells attach the surface electrodes. There is a direct correlation between the number of attaching cells and the value is redout by the RTCA. For migration and invasion, 20,000 cells per well were seeded in the upper chamber or in the 10% matrigel-coated chamber of cell invasion/migration (CIM) plates in serum-free medium. The bottom chambers of the CIM plates were filled with serum-containing medium to promote cell motility and migration. After seeding, the CIM plates were transferred into the RTCA-DP instrument for real-time readouts. Cell motility was assessed at 15-minute intervals for 30 hours. All experiments were repeated four times.
Immunofluorescent staining
Cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.3% TritonX-100 for 20 minutes and then blocked with 1% bovine serum albumin (Sigma-Aldrich) for 1 hour. For focal adhesion staining, the cells were incubated with mouse anti-vinculin antibody (Sigma-Aldrich) at 1:400 overnight at 4°C, followed by Cy3-conjugated secondary antibody (1:300, Invitrogen) for 1 hour at 25°C. Actin filaments were stained using TRITC-Phalloidin (1:250). The cells were mounted by using DAPI mounting medium (Zhongshan Jinqiao Biotech) and observed with a confocal laser scanning microscope with Leica TCS-SP system mounted on a Leica DMI6000 microscope (Leica). Images were collected in 512 × 512 pixel format. The cells were incubated without a primary antibody as negative control.
Immunohistochemistry
Sections (5 μm thick) were made from 4% paraformaldehyde-fixed and paraffin-embedded specimens. The sections were preincubated with pure methanol containing 3% H2O2 to inhibit endogenous peroxidase activity and then incubated in a primary antibody (anti-CK7, 1:400; or anti-ki67, 1:500, Abcam) overnight at 4°C, respectively. The secondary biotinylated goat anti-rabbit IgG was consequently applied. The immunoreactivity was visualized by using streptavidin–peroxidase along with 3, 3′-diaminobenzidine and 0.3% H2O2 as substrate. In control experiments, the primary antibodies were replaced by normal goat serum.
Western blot
Cells were lysed in buffer (50 mmol/L Tris-HCl, 150 mmol/L NaCl, 1% TritonX-100, 1% sodium deoxycholate, 0.1% SDS, 1 mmol/L PMSF). The protein concentration was determined by NanoDrop 2000c spectrophotometer (Thermo Scientific) using BCA Protein Assay Kit (Applygen Technologies). Whole-cell protein extracts (50 μg) were separated using SDS-PAGE and processed by Western blot analysis using anti- Keap1 (1:20,000, Sigma-Aldrich), vinculin (1:500, Santa Cruz Biotechnology) monoclonal antibodies, β-actin (1:2000, CoWin Biotech) and Myo9b (1:1000, cytoskeleton) polyclonal antibodies at 4°C overnight, followed by incubation with horseradish peroxidase–conjugated secondary antibodies (1:2000, Santa Cruz Biotechnology) for 1 hour. The protein bands were detected by ECL chemiluminescence and analyzed using the Bandscan analysis software (Sterling). A mouse anti-GAPDH (1:2,000, Applygen Technologies) antibody was used as an internal control. The intensity values were analyzed with ImageJ software (NIH).
RhoA G-LISA activation assay
RhoA activation was measured using luminescence-based G-LISA Activation kit (Cytoskeleton Inc.) according to the manufacturer's instructions. Briefly, the infected cells were harvested at 70% confluence and whole cell lysates were extracted. Protein concentrations were measured and balanced to a certain protein concentration between 1 and 2 mg/mL for the assay. Values were normalized by protein content using a colorimetric assay (Bio-Rad Laboratories) according to the manufacturer's recommendations.
Development of the tumor xenograft model
Forty-five male athymic nude mice BALB/c-nu/nu, ages 5 weeks, were purchased from the Laboratory Animal Center of Capital Medical University (Beijing, China). The animal experiments were approved by the Animal Research and Ethical Committee of Capital Medical University. For tumor xenograft establishments, A549 cells overexpressing green fluorescent protein or Keap1 (3 × 106 cells/100 μL) were subcutaneously injected into the right side of the axilla region of mice. Tumor and axillary lymph nodes were collected at 3, 4, 5, and 6 weeks, respectively, after which 5 tumors and 10 lymph nodes from five nude mice were examined each week. In addition, to avoid individual differences in mice, five mice were subcutaneously injected with A549 control cells and Keap1 overexpressing cells into the right and left sides, respectively. The mice were scanned with small animal magnetic resonance imaging (MRI) to observe tumor growth in vivo, and then sacrificed 6 weeks post-injection. Tumor size was measured twice a week using calipers, and was calculated as VT = Length × W2 × π/2.
MRI examination
Small animal MRI was performed with a 7T MR imaging system (PharmaScan70/16US) and surface coil of 35 mm in diameter. Animals were anesthetized using pentobarbital, and then placed in the coil to be scanned. The T2-weighted image sequence was scanned using the following parameters: repetition time/echo time, 2500/35 ms; imaging matrix, 256 × 192; field of view, 20 × 20 mm; slice thickness, 0.7 mm; intersection gap, 0 mm; and number of averages, 6.
Statistical analysis
The data are represented as the means of at least three independent experiments. Statistical analysis was performed using the unpaired Student t test and P value less than 0.05 was considered statistically significant.
Results
Effects of Keap1 on migration and invasion
A549, H460, and H1299 lung cancer cells were transfected by lentivirus with high efficiency infection, and Keap1 expression in the cells was verified by Western blot (Fig. 1A–C). The effects of Keap1 on the migration and invasion of cells were investigated by RTCA. Keap1 overexpression led to a significant reduction in the migration (Fig. 1D and E) and invasion (Fig. 1F and G) of A549 (P < 0.01), H460 (P < 0.01), and H1299 cells (P < 0.01). Serum-free culture medium was used to exclude the effects of proliferation on migration and invasion behaviors. These results demonstrate that the overexpression of Keap1 clearly inhibits the movement of NSCLC cells.
Effects of Keap1 on the migration and invasion of NSCLC cells. A and B, A549 cells were transfected by lentivirus with high efficiency infection, and Keap1 expression in cells was verified by Western blotting (C). Overexpression of Keap1 led to a significant reduction in the migration (D, E) and invasion (F, G) of A549, H460, and H1299 cells (P < 0.01). Right, The average slopes in certain periods of time from three parallel measurements.
Overexpression of Keap1 led to the formation of actin stress fibers in A549 cells
Actin cytoskeleton organization plays key roles in cell movement, and Keap1 is an actin-binding protein but it remains unclear if it can stabilize F-actin and affect cell behavior; therefore, we observed the reorganization of F-actin in different groups. Our results clearly showed that Keap1 was often colocalized with F-actin in the cytoplasm (Fig. 2A). Prominent F-actin bundles and abundant parallel stress fibers throughout the cytoplasm were distinctly seen in the Keap1-overexpressing cells, while control cells only showed lots of punctuated or short rod-like F-actin diffused throughout the cytoplasm (Fig. 2B). One observation field had obviously thicker and relatively stable F-actin stress fibers in Keap1-overexpressing cells compared with the nonexpressing cells (Fig. 2C). These data show that the overexpressed Keap1 in the cells evidently promotes the formation of stress fibers and reduces its reorganization.
Morphology and distribution of the F-actin cytoskeleton in Keap1-overexpressing A549 cells. A, Keap1 was colocalized with F-actin. B, Distribution of F-actin in Keap1-overexpressing cells and GFP control cells. C, In one observation field, thicker and well-aligned F-actin stress fibers could be observed in Keap1-overexpressing cells compared with nonexpressing cells. Green indicates Keap1 marked by GFP. Red indicates F-actin stained with TRITC-Phalloidin. The merged image is the enlarged picture shown in the box. Scale bar, 25 μm (A), 50 μm (B), 10 μm (C).
Keap1 inhibited turnover of the focal adhesion complex
It has been speculated that focal adhesions play a critical role in cell migration. In this study, the alterations of vinculin, a protein associated with cell–cell and cell–matrix junctions, in focal adhesions were detected. Clearly, the number and size of focal adhesions, mainly located in the cytoplasm, were significantly increased in the Keap1-overexpressing cells. In contrast, focal adhesions were tiny and sparsely arranged at the peripheral edge of the control cells (Fig. 3A). Moreover, the vinculin expression was significantly enhanced in the Keap1-overexpressing cells compared with control cells as shown by Western blot (Fig. 3B and C), suggesting that Keap1 not only effects on the maturation and distribution of focal adhesions, but also increases their protein levels. In addition, mature focal adhesion complexes attached to the ends of stress fibers in the Keap1-overexpressing cells, which may also stabilize the F-actin architecture (Fig. 3D). These results support that Keap1 can inhibit focal adhesion turnover, and enhance the formation of stress fibers to play an inhibited role of the cell motility.
Morphology and distribution of focal adhesion in Keap1-overexpressing cells. A, Keap1-induced changes of focal adhesion by vinculin immunostaining. Arrow points out tiny adhesion plaques along the plasma membrane of control cells, compared with large focal adhesion complexes dispersed in the cytoplasm of Keap1-overexpressing cells. B, Vinculin was upregulated in Keap1-overexpressing cells compared with wild-type and GFP control cells, as determined by Western blotting. C, Statistical data from three independent experiments are presented. D, Focal adhesion complexes bound the filament ends in Keap1-overexpressing cells. Scale bar, 50 μm.
Effects of Keap1 were mainly Myo9b–RhoA-dependent
RhoA is a key regulator of focal adhesion turnover and actin dynamics. As shown in Fig. 4, RhoA activity was markedly increased in the Keap1-overexpressing cells, but its expression was not obviously changed (Fig. 4A and B). Treating the cells with Y27632 (5 μmol/L, 24 hours), a Rho kinase inhibitor, resulted in actin depolymerization and the presence of very fine actin cables within the cytoplasm (Fig. 4C), which significantly enhanced cell migration (Fig. 4D). These results indicate that the decreased migration in Keap1 overexpression may mainly depend on RhoA activation. Previous studies have reported that class IX myosins (Myo9) are unconventional members of the myosin family in that their tail domain contains a RhoGTPase-activating protein. Of them, Myo9b, one member of the Myo9 family, specifically targets Rho protein and downregulates RhoA activity (26). Western blot showed that Myo9b expression was reduced in Keap1-overexpressing cells compared with the control. The decrease of Myo9b expression could be rescued by treatment with MG132 (2 μmol/L, 24 hours), a proteasome inhibitor (Fig. 5A). F-actin stress fibers in Keap1 overexpressing cells were reorganized to form actin network beneath the cell membrane after treatment with MG132 (Fig. 5B). These results indicate that Keap1 obviously enhances RhoA activity by promoting the ubiquitination-mediated proteasomal degradation of Myo9b.
Regulation of Keap1-dependent cell migration via a RhoA/ROCK-dependent mechanism. A and B, RhoA activity was markedly increased in Keap1-overexpressing cells, but its expression was not obviously changed. C, F-actin stress fibers were disrupted upon treatment with the inhibitor Y47632. D, Cell motility was rescued significantly after treatment with the Rho kinase inhibitorY27632 by RCTA. Data from three parallel experiments were used for statistical analyses. Scale bar, 50 μm.
Involvement of Myo9b in the regulation of Keap1 on F-actin reorganization. A, Expression of Myo9b was reduced in Keap1-overexpressing cells, which could be rescued by treatment with the MG132 proteasome inhibitor. B, F-actin was reorganized, becoming fewer and thinner and forming cortical fibers under the plasma membrane after MG132 treatment. Scale bar, 20 μm.
Tumor xenograft experiments
To examine the effects of Keap1 overexpression on tumor growth and metastasis in vivo, the mice with tumor xenografts were regularly checked for any sign of poor general health and stayed healthy until sacrifice. Tumor size was measured with calipers and the results are shown in Fig. 6. Keap1 overexpression cells clearly inhibited tumor growth and reduced the expression of Ki67, a proliferation marker (27), compared with the control group (Fig. 6A and B). MRI results also showed significant invasion and the rapid growth of tumors in the control group compared with Keap1-overexpressing tumors (Fig. 6C). Importantly, the tumor cells metastasis into lymph nodes was often observed (Fig. 6D), and occurred much earlier in control A549 tumors than in A549-Keap1 tumors (Table 1). Although these results might not be sufficient for evaluating tumor metastasis, it still reflects the motility of tumor cells in a certain extent.
Effects of Keap1 supression on the growth and metastasis of xenograft tumors. A, Tumor volume was measured every 2 days. Overexpressed Keap1 clearly inhibited tumor growth in vivo. B, Ki67 expression was significantly reduced in Keap1-overexpressing tumors compared with the control group. C, Subcutaneous edema, which might signify inflammatory infiltration, and a large area of necrosis were observed in the control group in T2-weighted magnetic resonance images, which were not prominent in the Keap1 group. The red arrow indicates necrosis, and the yellow arrow indicates inflammatory infiltration. D, Tumor cells were observed in lymph nodes by CK7 staining. Scale bar, 20 μm.
Lymph node metastasis in mouse models
Discussion
Keap1 is considered to be a tumor suppressor due to its ability to inhibit tumor progression (28). However, in the past years, Keap1 studies have mainly focused on antioxidative stress through the Keap1/Nrf2/ARE signaling pathway (29–31). It was reported that high methylation of CpG islands in the Keap1 promoter significantly downregulated its expression in NSCLC tissues and cells (32). Moreover, reducing Keap1 expression promoted breast cancer metastasis (33, 34). However, the mechanism underlying Keap1 inhibition of tumor metastasis and its effects on cell behaviors have remained unclear. In the present study, we demonstrated that overexpression of Keap1 not only promoted stress fiber formation but also inhibited focal adhesion turnover, in which the tumor cells exhibited prominent stress fibers and many large mature focal adhesions. The invasion and motility of NSCLC cells were significantly inhibited after Keap1 overexpression, which indicated that the Keap1 played vital roles in inhibiting NSCLC metastasis.
F-actin reorganization and focal adhesion turnover frequently play key roles in cell motility. Our study clearly saw the prominent dense stress fiber bundles distributed in parallel arrays throughout the cytoplasm of the Keap1 overexpression cells. Accordingly, cells enriched with stress fibers often showing slower movement in breast cancer cells (34, 35), which was also observed in our study, as cell motility was reduced when the F-actin cytoskeleton failed to reorganize or stabilize as stress fiber bundles. Furthermore, constant focal adhesion turnover was observed in the control cells; small new nascent adhesions formed at the leading edge, matured into focal adhesions, and then disassembling at the cell rear allowing for cell detachment. Generally, nascent adhesions are much smaller but transmit stronger forces than focal adhesions. Rapidly migrating cells constantly have many smaller adhesion clusters, whereas cells with large focal adhesions are typically either nonmigratory or move very slowly (36). In the current study, many larger mature focal adhesions were seen in the Keap1 overexpression cells than in the control, supporting that the Keap1 could inhibit the turnover of focal adhesions.
Regarding the mechanism of increased stress fiber bundles and mature focal adhesions in Keap1 overexpression cells, our results indicated that activity of the RhoA was clearly upregulated. Previous studies have reported that F-actin cytoskeleton reorganization and focal adhesion turnover are mediated by Rho GTPases signaling, which is a critical mechanism of cell migration during cancer metastasis (37). In this study, RhoA activity was significantly enhanced in the Keap1 overexpression cells compared with the control. Furthermore, migration deficiency in Keap1 overexpression cells could be partly rescued by treatment with the Rho-kinase inhibitor Y-27632. These results indicate that RhoA activity is important for inducing the stability of actin-stress fibers, which might contribute to the Keap1-mediated inhibition of cell migration.
It has been considered that myosinIXb (Myo9b), a Rho GTPase-activating protein, is essential for the negative regulation of Rho activity (38, 39). Keap1 acts as a substrate adaptor protein for the cullin 3-containing E3-ligase, which can degrade ubiquitinated proteins such as Nrf2. It is reported that Myo9b is highly expressed in lung cancer in which Keap1 is often downregulated (40). Because Keap1 colocalizes with F-actin where myo9b also bound. We therefore hypothesized that Keap1 might interact with Myo9b to ubiquitinate and degrade Myo9b, thereby upregulating RhoA activity. Actually, the present study demonstrated that Myo9b expression was clearly reduced in Keap1 overexpression cells and rescued after treatment with the MG132 proteasome inhibitor. Thus, it is reasonable to believe that Keap1 downregulate expression ofMyo9b protein and then, upregulate RhoA activity, thereby contributing to inhibition of the invasion and migration in NSCLC cells. This phenomenon was also observed in vivo where Keap1 inhibited lymph node metastasis in tumor xenografts. Additionally, Keap1 protein has five functional domains and its DGR domain often binds with actin proteins. This conformation not only explains the frequently observed colocalization of Keap1 with F-actin but also help to understand the role of Keap1 in directly stabilizing F-actin stress fibers and restraining the motility of NSCLC cells.
In conclusion, the present results indicate that Keap1 may stabilize the cytoskeleton by both directly binding to F-actin or by indirectly upregulating RhoA activity through decreasing expression of Myo9b protein, thereby inhibiting F-actin remodeling and focal adhesion turnover. Therefore, we propose that Keap1 may be a critical suppressor of metastatic progression of NSCLC, and increasing Keap1 or targeting its downstream molecules might provide potential therapeutic benefits for the treatment of patients with NSCLC.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Authors' Contributions
Conception and design: B. Wu, D. Zhou
Development of methodology: B. Wu, H. Sun, T. Sun
Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): S. Yang, H. Sun, T. Sun, Y. Wang, L. Xu
Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): B. Wu, S. Yang, Y. Wang, L. Xu, D. Zhou
Writing, review, and/or revision of the manuscript: B. Wu, D. Zhou
Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): H. Sun, T. Sun, F. Ji
Study supervision: B. Wu, F. Ji
Acknowledgments
This work was funded by the National Science Foundation of China (81101769, 81300285 and 31771332), Beijing Natural Science Foundation (7172021 and 5172008), Support Project of High-level Teachers in Beijing Municipal Universities in the Period of 13th Five-year Plan (IDHT20170516), and Science Foundation of Capital Medical University [17ZR09].
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
- Received October 10, 2017.
- Revision received November 12, 2017.
- Accepted December 7, 2017.
- ©2018 American Association for Cancer Research.
References
Volume 16, Issue 3
