ANKRD49 promotes the metastasis of NSCLC via activating JNK-ATF2/c-Jun-MMP-2/9 axis
BMC Cancer volume 23, Article number: 1108 (2023)
Ankyrin repeat domain 49 (ANKRD49) has been found to be highly expressed in multiple cancer including lung adenocarcinoma (LUAD) and lung squamous carcinoma (LUSC). However, the function of ANKRD49 in the pathogenesis of NSCLC still remains elusive. Previously, ANKRD49 has been demonstrated to promote the invasion and metastasis of A549 cells, a LUAD cell line, via activating the p38-ATF-2-MMP2/MMP9 pathways. Considering the heterogeneity of tumor cells, the function and mechanism of ANKRD49 in NSCLC need more NSCLC-originated cells to clarify.
Real-time qPCR was employed to test ANKRD49 expression levels in nine pairs of fresh NSCLC tissues and the corresponding adjacent normal tissues. The function of ANKRD49 was investigated using overexpression and RNA interference assays in lung adenocarcinoma cell line (NCI-H1299) and lung squamous carcinoma cell line (NCI-H1703) through gelatin zymography, cell counting kit-8, colony formation, wound healing, migration and invasion assays mmunoprecipitation was performed to in vitro. Immunoprecipitation was performed to test the interaction of c-Jun and ATF2. Chromatin immunoprecipitation was conducted to assess the transcriptional regulation of ATF2/c-Jun on MMP-2/9. Moreover, the tumorigenicity of ANKRD49 was evaluated in nude mice models and the involved signal molecular was also measured by immunohistochemical method.
We found that the levels of ANKRD49 in cancerous tissues were higher than those in adjacent normal tissues. in vitro assay showed that ANKRD49 promoted the migration and invasion of NCI-H1299 and NCI-H1703 cells via enhancing the levels of MMP-2 and MMP-9. Furthermore, ANKRD49 elevated phosphorylation of JNK and then activated c-Jun and ATF2 which interact in nucleus to promote the binding of ATF2:c-Jun with the promoter MMP-2 or MMP-9. In vivo assay showed that ANKRD49 promoted lung metastasis of injected-NSCLC cells and the high metastatic rate was positively correlated with the high expression of ANKRD49, MMP-2, MMP-9, p-JNK, p-c-Jun and p-ATF2.
The present study indicated that ANKRD49 accelerated the invasion and metastasis of NSCLC cells via JNK-mediated transcription activation of c-Jun and ATF2 which regulated the expression of MMP-2/MMP-9. The molecular mechanisms of ANKRD49’s function is different from those found in A549 cells. The current study is a supplement and improvement to the previous research.
Lung carcinoma is the most common cause of cancer-related deaths globally, with an estimated 1.6 million deaths annually . Non-small cell lung cancer (NSCLC) accounts for 85% of all cases , of which lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) are the most common subtypes . Notwithstanding unprecedented advances in the remedy of NSCLC, such as targeted therapy and immunotherapy, have been made in recent years, the long-term prognosis of NSCLC patients remains poor, with a 5-year survival rate of less than 15% [4, 5]. Therefore, it is essential to clarify the molecular pathogenesis of NSCLC and identify more precise prognostic markers and therapeutic targets.
The ankyrin repeat domain consists of 30 ~ 34 amino acid residues and mediates protein-protein interactions. ANKRD49 (ankyrin repeat domain 49) contains four ankyrin motifs. ANKRD49 is highly expressed in the testes and is involved in spermatogenesis [6, 7]. ANKRD49 also participates in the progression of malignant gliomas and gastric cancer in humans [8, 9]. Previously, we explored the expression pattern of ANKRD49 protein in NSCLC (80 cases of LUAD and 80 cases of LUSC) and found that the levels of ANKRD49 in cancer tissues were higher than those in tumor-adjacent normal tissues, its expression correlated with the TNM (tumor-node-metastasis) stage, distal metastasis, lymph node metastasis and differentiation . Moreover, patients with higher ANKRD49 showed lower OS (overall survival) rate and higher ANKRD49 expression in lung tissues may serve as an independent prognostic marker for NSCLC patients [10, 11]. Howbeit, the function and underlying mechanisms of ANKRD49 in NSCLC has not yet been fully elucidated.
ANKRD49 has been demonstrated to promote the invasion and metastasis of A549 cells, a lung adenocarcinoma cell line, via activating the p38/ATF-2 signaling pathway and then elevating the levels of MMP2/MMP9 in our previous study . Considering the heterogeneity of tumor cells, the function and mechanism of ANKRD49 in NSCLC need more NSCLC-originated cells to clarify. Therefore, in the present study, another LUAD cell line (NCI-H1299) and a LUSC cell line (NCI-H1703) were selected and our findings illustrated that ANKRD49 accelerated the migration and invasion of NCI-H1299 and NCI-H1703 cells via the JNK-ATF2/c-Jun-MMP-2/MMP-9 pathway, unlike its function in A549 cells.
Materials and methods
Human tissue samples
Nine pairs of fresh NSCLC tissues and the corresponding adjacent normal tissues were collected from patients who underwent surgery at the First Hospital of Shanxi Medical University from October 2020 to December 2020. No patients in this study received other treatment before surgery.
Cell lines and cells culture
The human bronchial epithelial cell line (HBEC) and NSCLC cell lines H1299, A549, H446, H460, Calu-3, H1703, sk-MES-1 were obtained from the Cell Culture Center of the Chinese Academy of Medical Sciences (Beijing, China). These NSCLC cell lines were cultured in Advanced RPMI 1640 medium (Seven, Beijing, China) supplemented with 10% fetal bovine serum (Every Green, Zhejiang, China) and incubated at 37 °C in 5% CO2.
Establishment of ANKRD49 stably expressed cell lines
H1299 and H1703 cells were infected with lentivirus-5 ANKRD49 or lentivirus-3 ANKRD49-shRNA and corresponding vectors (Gene Pharma, Shanghai, China) according to the manufacturer’s protocols to construct the stable ANKRD49 overexpression (ANKRD49-OE) or ANKRD49 knockdown (ANKRD49-sh) groups. Meanwhile, the controls for overexpression (LV5) and knockdown (LV3) were also established. After infection of lentivirus, cells were incubated for more than 48 h and puromycin (2 µg/ml) was added to screen the stable cells. LV3 and ANKRD49-sh sequences were listed in Supplementary Table 1.
Reagents and antibodies
Puromycin (P816466) was purchased from MACKLIN (Shanghai, China). Anisomycin (GC11559) was purchased from GLPBIO (Shanghai, China). The p38 mitogen-activated protein kinase (MAPK) inhibitor (HY-10,256, SB203580) and JNK inhibitor (HY-12,041, SP600125) were purchased from MedChemExpress (New Jersey, USA). MMPs inhibitor (SF4180, ilomastat) was purchased from Beyotime Biotechnology (Shanghai, China). Rabbit anti-ANKRD49 primary antibody (Proteintech, Cat# 25034-1-AP, RRID:AB_2879860), rabbit anti-MMP-2 polyclonal antibody (Cat# bs-4599R, RRID:AB_11083963), rabbit anti-MMP-9 polyclonal antibody (Cat# bs-0397R, RRID:AB_10853038), rabbit anti-c-Jun polyclonal antibody (Cat# bs-0670R, RRID:AB_10857880), rabbit anti-p-ATF2 polyclonal antibody (Cat# bs-3033R, RRID:AB_10883830) and Alexa Fluor® 488-conjugated goat anti-rabbit IgG (Cat# bs-0295G-A488, RRID:AB_10893781) were obtained from Bioss Biotechnology (Beijing, China). The mouse anti-p-c-Jun monoclonal antibody (Cat# 558,036, RRID: AB_2249448) was obtained from BD Biosciences (New Jersey, USA). Rabbit anti-p38 monoclonal antibody (Cat# 8690, RRID: AB_10999090), rabbit anti-p-p38 monoclonal antibody (Cat# 4511, RRID: AB_2139682), rabbit anti-ERK monoclonal antibody (Cat# 4695, RRID: AB_390779), rabbit anti-p-ERK monoclonal antibody (Cat# 4376, RRID: AB_331772), rabbit anti-SAPK/JNK monoclonal antibody (Cat# 9258, RRID: AB_2141027), and rabbit anti-p-JNK monoclonal antibody (Cat# 4668, RRID: AB_823588) were purchased from Cell Signaling Technology (Danvers, MA, USA). Rabbit anti-ATF2 polyclonal antibody (Cat# BS1022, RRID: AB_1664115), rabbit anti-β-actin polyclonal antibody (Cat# AP0060, RRID: AB_2797445), rabbit anti-GAPDH polyclonal antibody (Cat# AP0063, RRID: AB_2651132), rabbit anti-Histone-H3 polyclonal antibody (Cat# BS7675), rabbit anti-PARP polyclonal antibody (Cat# BS7190), and rabbit anti-β-tubulin polyclonal antibody (Cat# AP0064, RRID: AB_2797447) were acquired from Bioworld (Minnesota, USA). Rabbit anti-ATF2 polyclonal antibodies (Cat# D155200) and rabbit anti-p-ATF2 polyclonal antibodies (Cat# D155010) were purchased from Sangon Biotech (Shanghai, China). Anti-rabbit IgG (H + L) (Cat# BA1050, RRID: AB_2904507) and anti-mouse IgG (H + L) (Cat# BA1054, RRID: AB_2734136) were purchased from Boster Biological Technology (Wuhan, China). Dylight 649 and goat anti-mouse IgG (Cat# 610-143-002, RRID: AB_11182582) were obtained from Rockland (Philadelphia, USA).
Total protein from cells or tissues was extracted using SDS lysis buffer containing 2% SDS, 10% glycerol, and 50 mM Tris-HCl (pH 6.8), and quantified using the Enhanced BCA Protein Assay Kit (Beyotime Biotechnology). Proteins (30 µg) were loaded onto a 12% SDS-PAGE gel, separated, and transferred onto polyvinylidene fluoride (PVDF) membranes. The membranes were blocked using 5% skimmed milk, followed by probing with the indicated primary antibodies at 4 °C overnight, and then incubated with an anti-rabbit/mouse HRP-conjugated IgG at room temperature for 1 h. Blots were developed using the ECL kit (Seven, Beijing, China) and detected using the ChemiDoc imaging system (Bio-Rad, California, USA).
Real-time qPCR analyses
Total RNA from NSCLC cells, nine pairs of human NSCLC tissues, and matched adjacent normal tissues were prepared using the Total RNA Extraction Kit (Promega, Madison, WI, USA) following the manufacturer’s instructions. RT-qPCR was performed using the RT-PCR Kit (Seven, Beijing, China) on the Quant Studio 3 (Thermo Fisher Scientific, Massachusetts, USA), according to the manufacturer’s procedures. All the primers are listed in Supplemental Table 2. β-actin was used as the control, and the relative mRNA expression was defined using the 2 − ΔΔCt method.
MMP-2 and MMP-9 activities were evaluated using gelatin zymography. 5 × 105 cells were pretreated with serum-free medium for 48 h. The medium was collected, centrifuged at 1,500× g for 15 min at 4 °C, and mixed with non-reducing sample buffer for electrophoresis on a polyacrylamide gel containing 0.1% (w/v) gelatin. Proteolysis was assessed as a white zone in the dark blue fields. The 62 kDa and 92 kDa bands corresponded to active MMP-2 and MMP-9, respectively. The gelatinase activity of MMP-2 and MMP-9 was illustrated via the area of the clear zone in the dark blue gel and analyzed using ImageJ analysis software (National Institute of Health, Maryland, USA).
Cell counting kit-8 (CCK-8) assay
The CCK-8 assay was performed to evaluate cellular proliferation, according to the manufacturer’s protocols. Briefly, ANKRD49-OE or LV5 H1299 cells, ANKRD49-sh, or LV3 H1703 cells were cultured in 96-well plates at a density of 1 × 103 cells per well in a 5% CO2 incubator at 37 °C. Next, 100 µl serum-free medium containing 10 µl CCK-8 reagent (Seven, Beijing, China) was added to each well, followed by another 1 h incubation. The absorbance of the cells was measured at 450 nm using a microplate reader (Molecular Devices, California, USA).
Colony formation assay
ANKRD49-OE or LV5 H1299 cells, ANKRD49-sh, or LV3 H1703 cells were plated in six-well plates at a density of 5 × 102 cells/well and cultured at 37 °C for 1 week. After washing with PBS, the cells were fixed with methanol and stained with 0.1% crystal violet solution. The colonies were photographed and quantified using a light microscope (Eclipse Ts2; Nikon, Tokyo, Japan).
Wound healing assay
Cells (2 × 105) were seeded into six-well plates and cultured for 24 h. Wounds in the confluent cells were created using a 200 µl pipette tip, followed by washing the cells with PBS to remove any floating cells. Next, complete medium was added and the wound margins were photographed. Subsequently, wound healing was photographed at 24 and 48 h using a Nikon photomicroscope (Eclipse Ts2, Nikon, Tokyo, Japan). Migratory ability was calculated by measuring the distance between different groups of cells simultaneously.
Migration and invasion assays
Cells (2 × 104) were seeded into the upper chamber of 24-well inserts (8.0 μm, Corning, California, USA). The upper chamber was pre-coated with Matrigel (BD Biosciences, California, USA), and a medium containing 10% FBS was added to the lower chamber for the invasion assay. After incubation at 37 °C for 24 h, the remaining cells in the upper chamber were scraped off and the cells on the lower side of the chamber were fixed, stained, and viewed using a Nikon photomicroscope (Eclipse Ts2). Data were analyzed by counting at least five random fields.
Cytoplasmic and nuclear protein extraction
ANKRD49-OE or LV5 H1299 cells were plated in 10 cm plates at a density of 5 × 106 cells/plate and cultured at 37 °C for 2 days. The subcellular proteins in ANKRD49-OE or LV5 H1299 cells were prepared using a subcellular structure cytoplasm and nucleus extraction kit (Bioss Biotechnology). In brief, samples were centrifuged at 750 × g for 5 min at 4 °C. The supernatant was collected and centrifuged at 3400, × g, 10 min (cytoplasmic fraction). This process was repeated four times to remove nuclear contamination. The pellet was washed with fractionation buffer and centrifuged at 750 × g, 5 min to remove cytoplasmic contamination at least three times. The nuclear/membrane fraction was dissolved in RIPA buffer. The protein samples (20 µg) were subjected to Western blot with p-ATF2 or p-c-Jun antibodies.
H1299 cells were transfected with pMSCVpuro-ANKRD49 or the vector for 48 h, and 5 × 104 cells were seeded onto sterile coverslips in a twelve-well plate for another 24 h. The medium was removed, and cells were fixed with 4% paraformaldehyde and then permeabilized with 0.01% Triton X-100 at room temperature for 10 min. Cells were incubated with anti-p-ATF2 antibody or anti-p-c-Jun antibody at 4 °C overnight, and then probed with Alexa Fluor® 488-conjugated goat anti-rabbit IgG or Dylight 649 goat anti-mouse IgG at room temperature for 1 h in darkness. Nuclei were stained with DAPI (Beyotime) for 5 min, and fluorescence signals were observed using a fluorescence microscope (Eclipse Ts2).
Nucleus proteins of the ANKRD49-OE or LV5 H1299 cells (1 × 106) were extracted and 25 µg of them were precleared using Protein A/G Agarose (Santa Cruz, California, USA). Subsequently, 2 µl rabbit anti-ATF2 or normal rabbit IgG was added to the lysates and incubated at 4 °C for 12 h. Next, 20 µl of Protein A/G PLUS Agarose was mixed into the lysates and incubated at 4 °C overnight. Agarose was washed three times using wash buffer, and the bound proteins were separated by SDS-PAGE.
Chromatin immunoprecipitation assay
The chromatin immunoprecipitation (CHIP) assay was conducted in accordance with the manufacturer’s protocols (Beyotime Biotechnology). The binding site of ATF2/c-Jun in the MMP-2 or MMP-9 promoter region and the position of the CHIP primers are illustrated in Supplementary Fig. S1. Chromatin solutions were sonicated and incubated with anti-p-ATF2, anti-p-c-Jun, or control IgG at 4 °C for 12 h. DNA-protein cross-links were reversed, and chromatin DNA was subsequently purified and subjected to PCR analysis. Primers for amplifying the MMP-2 or MMP-9 promoter, which contains the binding site of ATF2/c-Jun, are listed in Supplementary Table 3. The immunoprecipitated DNA was analyzed by qPCR. The qPCR products were resolved on a 1.2% agarose gel and visualized by nucleic acid dye staining.
Mouse xenograft and metastasis models
Ten female BALB/c nude mice and ten male BALB/c nude mice (5~6-week-old) were obtained from the Experimental Animal Center of Shanxi Medical University. The mice were housed under SPF conditions with a 12 h light-dark cycle and ad libitum access to food and water at 23 °C with 60% humidity. Female mice were randomly allocated to either LV5 (n = 5) or ANKRD49-OE H1299 cells (n = 5). Male mice were randomly allocated to either LV3 (n = 5) or ANKRD49-sh H1703 cells (n = 5). One week of acclimation, 4 × 106 cells in 200 µl saline were injected into the lateral tail veins of the nude mice. After the injection, the health of the mice was monitored daily. After 4 weeks, the mice were sacrificed, and whole lungs were collected to count the metastatic nodules on the surface. Lung tissues were fixed in 10% paraformaldehyde for further hematoxylin-eosin (H&E) staining and IHC analysis. All animal experiments were approved by the Animal Ethics Committee of the Shanxi Medical University (approval number SYDL-2020016) and was performed according to the procedures that complied with the ARRIVE guidelines.
Five micrometer-thick sections of formalin-fixed paraffin-embedded lung tissues from nude mice were prepared for IHC staining. Slides were deparaffinized, rehydrated, and microwaved for antigen retrieval in 0.01 M, PH 6.0 sodium citrate buffer. Endogenous peroxidase activity was blocked using 3% H2O2. Subsequently, sections were incubated with the corresponding primary antibody (1:100) for 45 min at room temperature. Diaminobenzidine (DAB) (Boster, Wuhan, China) was used to visualize peroxidase activity. The immunostaining intensity of ANKRD49, NAPSA, NKX2-1, MMP-2, MMP-9, p63, p-JNK, p-c-Jun, and p-ATF2 was calculated according to the published H-score method: H-score value = (unstained tumor cells) % × 0 + (weakly stained tumor cells) % × 1 + (moderately stained tumor cells) % × 2 + (strongly stained tumor cells) % × 3.
Correlations between the level of ANKRD49 and the levels of MMP-2, MMP-9, p-JNK, p-ATF2, and p-c-Jun were analyzed using Pearson’s correlation coefficient. A strong correlation was defined as an r-value of ≥ 0.5. SPSS18.0 (IBM, Inc) and GraphPad Prism7.0 (GraphPad Software Inc.) software were used to analyze the data. The analyses were repeated in triplicate. Quantitative data are expressed as the mean ± standard deviation (SD) and analyzed using a Student’s t‑test or one‑way analysis of variance followed by Tukey’s post hoc test using GraphPad Prism software. A two‑sided P‑value < 0.05 was considered to indicate a statistically significant difference.
ANKRD49 is highly expressed in NSCLC
To investigate the expression pattern of ANKRD49 in fresh tissues from NSCLC, nine fresh tumor tissues and adjacent normal tissues (6 cases of LUAD and 3 cases of LUSC) were collected and the ANKRD49 mRNA was measured using RT-qPCR. The data showed that the expression of ANKRD49 mRNA in tumor tissues was significantly higher than that in adjacent normal tissues (Fig. 1A). Next, the expression of ANKRD49 mRNA in human bronchial epithelial cell line (HBEC), as well as in five LUAD lines (H1299, A549, H446, H460, Calu-3) and two LUSC lines (H1703, sk-MES-1) was assessed by RT-qPCR. The results demonstrated that the expression of ANKRD49 was obviously higher in H1299, A549, H446, H1703, and sk-MES-1 cells than in the human bronchial epithelial cell line (Fig. 1B). Accordingly, H1299 cell line was selected for ANKRD49 overexpression assay, while H1703 cell line was opted for ANKRD49 knockdown analysis.
ANKRD49 promotes the migration and invasion of H1299 cells
To investigate the function of ANKRD49 in LUAD cells, we established stable ANKRD49-OE or ANKRD49-sh H1299 cells, as well as their respective control cells LV5 and LV3. RT-qPCR and Western blot assays showed that ANKRD49-OE and ANKRD49-sh H1299 cells were constructed (Fig. 2A, B). CCK-8 and colony formation assays revealed that ANKRD49 had no effect on H1299 cell proliferation (Supplementary Fig. S2). Wound healing, transwell migration, and invasion assays illustrated that ANKRD49-OE markedly enhanced the migration and invasion of H1299 cells compared to the LV5 group, and the opposite results were observed in the ANKRD49-sh cells compared to the LV3 group (Fig. 2C-F). Herein, we wanted to know whether endogenous levels of ANKRD49 correlated with differential endogenous migration/invasion potential, wound healing, transwell migration and invasion assays were performed using H1703 cells (higher expression of ANKRD49) and H1299 cells (lower expression of ANKRD49). The results exhibited that H1703 cells had the higher capability in cellular migration and invasion than those in H1299 cells (Supplementary Fig. S3). Taken together, these data demonstrated that ANKRD49 accelerated the migration and invasion of H1299 and 1703 cells.
ANKRD49 upregulates MMP-2/MMP-9 expression by activating the MAPK pathway in H1299 cells
The high expression of MMP-2 and MMP-9 in cancerous tissues has been correlated with tumor cell matrix degradation, invasion, and migration [12,13,14]. To uncover the molecular basis of ANKRD49 on invasion and migration, the expression of MMP-2 and MMP-9 was tested when ANKRD49 was overexpressed or downregulated by RT-qPCR and Western blot assays. The results revealed that MMP-2 and MMP-9 levels were elevated in ANKRD49-OE and attenuated in ANKRD49-sh H1299 cells compared with the LV5 and LV3 groups, respectively (Fig. 3A, B). Next, the gelatinase activities of MMP-2 and MMP-9 were markedly boosted in ANKRD49-OE H1299 cells and declined in ANKRD49-sh H1299 cells, compared with those in the LV5 and LV3 groups, respectively (Fig. 3C, D). In order to confirm whether the effect of ANKRD49 was dependent on MMP-2 and MMP-9, ANKRD49-OE H1299 cells were pretreated with 10 µM ilomastat (MMP inhibitor) for 1 h. and the wound healing assay displayed that inhibition of MMP-2/MMP-9 attenuated the migration and invasion of H1299 cells (Fig. 3E, F).
It is well established that mitogen-activated protein kinases (MAPKs), including p38 MAPK, ERK, and JNK, are involved in tumor metastasis and invasion [15,16,17]. As illustrated in Fig. 4A, the levels of p-p38 and p-JNK were obviously augmented in ANKRD49-OE and decreased in ANKRD49-sh H1299 cells compared with the LV5 and LV3 groups, respectively, while the levels of p-ERK were not altered. Next, p38 MAPK inhibitor (10µM SB203580), JNK inhibitor (10µM SP600125), or DMSO (10 µL) was used to pretreat ANKRD49-OE H1299 cells for 1 h. We found that SP600125 significantly decreased MMP-2/MMP-9 levels compared to DMSO-treated cells, whereas SB203580 had no effect on MMP-2/MMP-9 expression (Fig. 4B, C). Besides, the wound healing assay also exhibited that inhibition of JNK abated the migration and invasion of ANKRD49-OE H1299 cells, whereas inhibition of p38 had no effect on migration and invasion of ANKRD49-OE H1299 cells (Fig. 4D, E). To explore the specific role of JNK on MMP-2/MMP-9 expression, p-ATF2 and p-c-Jun, which are regulatory targets of JNK , were analyzed by Western blot. As shown in Fig. 4F, the levels of p-ATF2 and p-c-Jun were both boosted in ANKRD49-OE and weakened in ANKRD49-sh H1299 cells compared with the LV5 and LV3 groups, respectively. Since ATF2 also has been reported to be regulated by p38 MAPK , in order to clarify whether p38 MAPK was involved in the regulation of ATF2 in ANKRD49-OE settings, SP600125 or SB203580 was used and p-ATF2 was analyzed. The results illustrated that SP600125 decreased the level of p-ATF2, but SB203580 did not (Fig. 4G, H), indicating that p38 MAPK didn’t participate in the regulation of ATF2 in ANKRD49-OE H1299 cells. Overall, these data demonstrate that ANKRD49 promotes the invasion and migration of H1299 cells by enhancing MMP-2/MMP-9 expression mediated by the JNK but not p38 MAPK pathway.
ANKRD49 activates ATF2/c-Jun transcription factor as a heterodimer to regulate MMP-2/MMP-9 expression in H1299 cells
ATF2 and c-Jun belong to the AP-1 family , translocate into the nucleus, and regulate the expression of target genes, including MMPs [21, 22]. Accordingly, the nuclear distribution of p-ATF2 and p-c-Jun was measured. The results showed that the nuclear levels of ATF2 and c-Jun as well as their phosphorylation were significantly enhanced in ANKRD49-OE H1299 cells compared to those in the LV5 group (Fig. 5A, B). It is widely acknowledged that ATF2 contains a nuclear export signal (NES) in its leucine zipper region and two nuclear localization signals (NLS) in its basic region, leading to continuous shuttling between the nucleus and the cytoplasm . It is essential for ATF2’s transcriptional activation that ATF2 dimerizes with c-Jun in the nucleus which prevents the export of ATF2 . Co-immunoprecipitation assay revealed that ANKRD49 promoted the interaction between p-ATF2 and p-c-Jun in the nucleus (Fig. 5C). In addition, immunofluorescence assay also showed that ANKRD49 promoted the nuclear co-localization of p-ATF2 and p-c-Jun (Fig. 5D). Furthermore, chromatin immunoprecipitation assay was conducted to analyze the binding ability of ATF2 or c-Jun to the MMP-2 or MMP-9 promoter region. Our results illustrated that ANKRD49 enhanced the binding of ATF2 or c-Jun to the MMP-2 or MMP-9 promoter region (Fig. 5E-G). Taken together, these findings suggest that ANKRD49 activates ATF2/c-Jun transcription factor as a heterodimer to regulate MMP-2/MMP-9 expression in H1299 cells.
ANKRD49 promotes migration and invasion of H1703 cells via MMP-2/MMP-9 mediated by JNK-ATF2/c-Jun pathway
Furthermore, the function of ANKRD49 in LUSC was investigated by establishing stable ANKRD49 knockdown (ANKRD49-sh) H1703 cells and a corresponding control LV3. RT-qPCR and Western blot assays demonstrated that ANKRD49-sh H1703 cells were successfully prepared (Fig. 6A, B). CCK-8 and colony formation assays revealed that ANKRD49 had no effect on H1703 cells’ proliferation (Supplementary Fig. S4). Wound healing, transwell migration, and invasion tests exhibited that ANKRD49-sh dramatically attenuated the migration and invasion of H1703 cells compared to the LV3 group (Fig. 6C-F). Additionally, we found that downregulation of ANKRD49 diminished both the expression and activity of MMP-2 and MMP-9 (Fig. 7A-D). Similarly, downregulation of ANKRD49 reduced the levels of p-JNK, p-p38, p-ATF2, and p-c-Jun in H1703 cells (Fig. 7E, F). To further confirm whether the effect of ANKRD49 on the MMP-2/MMP-9 levels in H1703 cells was dependent on JNK pathway just like it’s function in H1299cells, Anisomycin (20µM), an activator of JNK and p38 MAPK  was used to pretreat ANKRD49-sh H1703 cells for 1 h, followed by administration with SB203580, SP600125 or DMSO for another 24 h. We found that Anisomycin significantly augmented MMP-2/MMP-9 levels in SB203580-treated cells, whereas Anisomycin had no effect on MMP-2/MMP-9 expression in SP600125-treated cells (Fig. 7G, H). These data suggest that ANKRD49 promotes the invasion and migration of H1703 cells by enhancing MMP-2/MMP-9 expression mediated by the JNK rather than p38 MAPK pathway.
ANKRD49 potentiates migration and invasion of H1299 and H1703 cells in nude mice
To further explore the effect of ANKRD49 on the migration of NSCLC cells, ANKRD49-OE or LV5 H1299 cells and ANKRD49-sh or LV3 H1703 cells were injected intravenously into the tail of nude mice and maintained for 4 weeks. The results appeared that ANKRD49-OE enhanced the incidence of lung metastasis and the number of metastatic foci (Fig. 8A, B). Downregulation of ANKRD49 substantially reduced the incidence of lung metastasis and number of metastatic foci (Fig. 9A, B). Images of the mouse lungs are shown in Supplementary Fig. S5. HE staining also illustrated that more tumor cells appeared in perivascular and peribronchial regions in lung tissues from mice injected with ANKRD49-OE H1299 cells than in control mice (Fig. 8C). Nevertheless, the distribution of tumor cells in the lung tissues of mice injected with ANKRD49-sh or LV3 showed the opposite trend (Fig. 9C).
NAPSA and NKX2-1 are markers for LUAD [26,27,28], whereas p63 is a marker for LUSC . Immunohistochemical staining illustrated that the distribution of ANKRD49-positive cells as well as NAPSA-positive, NKX2-1-positive, or p63-positive cells was consistent with the results of HE staining (Figs. 8D and 9D). In addition, immunohistochemical staining showed that the levels of ANKRD49, MMP-2, MMP-9, p-JNK, p-c-Jun, and p-ATF2 were elevated in lung tissues from mice injected with ANKRD49-OE H1299 cells (Fig. 8E-G), whereas their levels were decreased in lung tissues of mice injected with ANKRD49-sh H1703 cells (Fig. 9E-G), compared to control mice. Correlation analysis displayed that the metastatic rates of the tumor cells were positively correlated with the expression of ANKRD49, MMP-2, MMP-9, p-JNK, p-c-Jun or p-ATF2 (Supplementary Fig. S6). In addition, MMP-2, MMP-9, p-JNK, p-ATF2, and p-c-Jun levels were positively correlated with the levels of ANKRD49 (Supplementary Fig. S7). In summary, ANKRD49 accelerated the invasion and metastasis of NSCLC cells via JNK-mediated transcription activation of c-Jun and ATF2 which regulated the expression of MMP-2/MMP-9.
The ANKRD49 protein contains four ankyrin repeat motifs involved in multiple protein-protein interactions in diverse life activities . It has been documented that ANKRD49 is highly expressed in several carcinomas, including gastric cancer and malignant gliomas. As an oncogene in malignant gliomas, ANKRD49 reduces cellular apoptosis and facilitates cell cycle progression to promote the proliferation of glioma cells. ANKRD49 can be an attractive therapeutic target for malignant glioma. . It also has been documented that ANKRD49 may serve as an independent prognostic indicator of gastric cancer as it is highly expressed . Similar results from a previous study showing ANKRD49, as one of the four gene signature from NCI-60 cell line, had a strong prediction value for non-small-cell lung cancer . Our previous study using microarray analysis combined with IHC staining of tissue from 160 NSCLC patients, including 80 LUAD cases and 80 LUSC cases, showed that ANKRD49 is highly expressed in cancerous lung tissues and correlates with lymph node metastasis, distal metastasis, TNM stage, and differentiation in NSCLC patients. Furthermore, higher ANKRD49 expression was associated with poor OS in patients with NSCLC . Another previous study from us showed that ANKRD49 promoted the invasion and migration of A549 cells via a P38 MAPK/ATF-2/MMPs signaling pathway in vitro and in vivo . These data implied that ANKRD49 participates in the occurrence and development of NSCLC.
Previously, we used A549 cells, a LUAD cell line, to explore the function of ANKRD49. We found that ANKRD49 promoted the metastasis of A549 cells via upregulation of MMP- 2 and MMP- 9 in a P38/ATF- 2 pathway- dependent manner . In consideration of different subtypes of NSCLC as well as heterogeneity of tumor cells, the function of ANKRD49 in NSCLC progression need further clarification. Herein, we used a lentivirus-mediated overexpression vector to upregulate ANKRD49 expression in H1299 cells, another LUAD cell line, and found that ANKRD49 potentiated the invasion and metastasis of H1299 cells. Furthermore, we utilized lentivirus-mediated shRNA, a loss-of-function strategy, to downregulate ANKRD49 expression in H1299 and H1703 cells (a LUSC cell line), and discovered that knockdown of ANKRD49 suppressed the invasion and migration of these cancer cells. Importantly, we also found that differential endogenous levels of ANKRD49 in H1299 and H1703 cells also have correspondingly differential migration and invasion (supplemental Fig. S3). The migration and invasion of cancer cells into surrounding tissues and vasculature are the crucial initial step in tumor metastasis, enabling the tumor to colonize distant organs . It is widely acknowledged that metastasis is a key cause of cancer-related death, and cell migration is a sophisticated multistep process . During this multistep process, degradation of the extracellular matrix (ECM) is an early event in tumor metastasis . MMPs belong to a family of proteolytic enzymes with many physiological roles, including ECM modification, which accelerates cell migration . Among the MMP family, MMP-2 and MMP-9 are documented as substrate-specific gelatinases that are pivotal in ECM degradation  and are advantageous for invasion and metastasis of tumor cells, leading to tumorigenesis . Our study demonstrated that ANKRD49 promotes the expression and activity of MMP-2 and MMP-9, which enhances the migration and invasion of NSCLC cells.
The JNK and p38 MAPK signaling pathways are characterized by kinase cascades in cancer biology and play a central role in the carcinogenesis and maintenance of cancers [37, 38]. JNK and p38 MAPK have been identified as crucial mediators of migration and invasion in various tumors [39, 40]. In this study, the phosphorylation of JNK and p38 was elevated in ANKRD49 ectopic expressed H1299 cells and decreased in ANKRD49 downregulated H1299 and H1703 cells. So, we speculated that the JNK and p38 MAPK pathways were involved in ANKRD49-induced cellular migration and invasion. However, unlike the JNK inhibitor, the p38 MAPK inhibitor did not induce a decline in the levels of MMP-2 and MMP-9. These data demonstrate that the JNK signaling pathway participates in ANKRD49-induced migration and invasion of H1299 and H1703 cells. Activated p38 signaling pathway may or may not contribute to tumor immunity  rather than metastasis in current settings. The present results were inconsistent with those in A549 cells, a possible reason was that these three types of cell lines have diverse genetic and histological backgrounds. A549 cells have been tested for the KRAS and KEAP1 (kelch like ECH associated protein 1) missense mutation, STK11 (serine/threonine kinase 11) nonsense mutation, wild type p53 expression, while H1299 cells have a homozygous partial deletion of the p53 protein, and lack expression of p53 protein . Of course, how these different genetic backgrounds affect p38 signaling pathway remains to be further studied.
Subsequently, we sought to understand how JNK regulates MMP-2 and MMP-9 expression. MMP-2 or MMP-9 transcription is reportedly regulated by various transcription factors, including AP-1, SP-1, NF-κB, and CREB . ATF2 and c-Jun are components of the dimeric transcription factor AP-1 and play crucial regulatory roles in many biological processes . Both ATF2 and c-Jun can be phosphorylated by JNK and subsequently translocated into the nucleus to function as transcription factors . The phosphorylation of ATF2 and c-Jun, as well as their nuclear distribution, were elevated in ANKRD49 overexpressed H1299 cells, and downregulation of ANKRD49 inhibited this tendency. It has been reported that in the absence of c-Jun, the monomers of ATF2 continuously shuttle between the nucleus and cytoplasm because it contains a nuclear export signal (NES) and two nuclear localization sequences (NLS), while the homodimers of ATF2 are predominantly distributed in the cytoplasm . To function as a transcription factor, ATF2 needs to form a heterodimer with c-Jun, which masks NES and prevents ATF2 nuclear export . Herein, we found that ANKRD49 boosted the interaction between ATF2 and c-Jun in the nucleus through Co-IP and immunofluorescence staining assays. It is well established that the promoter of MMP-2/MMP-9 contains the binding site of c-Jun , yet the binding sites of ATF2 in the promoter region of MMP-2 and MMP-9 have not been revealed. In the present study, a CHIP assay was carried out, and it was discovered that ATF2 binding sites exist in -1795~-1788 of the MMP-2 promoter region and − 2042~-2395 of the MMP-9 promoter region. ANKRD49 facilitated binding of ATF2 to the promoter regions of MMP-2 and MMP-9. In the end, our in vitro tests confirmed the ANKRD49’s function. Consequently, we conclude that ANKRD49 mediates MMP-2/MMP-9 via ATF2/c-Jun heterodimers, which are activated by JNK to promote the migration and invasion of NSCLC cells. Of course, apart from MMPs and MAPKs, other mechanisms such as epithelial-to-mesenchymal transition (EMT), angiogenesis or the stemness of tumor cells may be invovled in ANKRD49’s function on migration and invasion of NSCLC cells. Therefore, a more comprehensive mechanism will be investigated in our future work.
Interestingly, a recent study demonstrated that ANKRD49 expression is positively correlated to glioma grade and manifests a significant prognosis difference between glioma samples and non-glioma samples, suggesting that ANKRD49 may serve as a prognosis predictor . Similar results from our previous study showed that ANKRD49 protein in LUSC was elevated and correlated positively with the tumor node-metastasis stage, lymph node metastasis, distal metastasis, and differentiation. Patients with higher ANKRD49 had lower overall survival rate and higher ANKRD49 expression in lung tissues may be used as an independent prognostic marker for LUSC patients . These results indicated that ANKRD49 could be a potential pan-cancer predictor. Our study defines a mechanism for ANKRD49 in acceleration of invasion and metastasis of NSCLC cells (Fig. 10). In turn, inactivating the JNK-ATF2/c-Jun pathways inhibit NSCLC metastasis. Therefore, ANKRD49 may serve as a potential anti-metastatic therapeutic target and a novel prognostic biomarker for NSCLC.
The present study has several unaddressed issues. The role of activated p38 MAPK in ANKRD49-OE H1299 or ANKRD49-sh H1703 cells remains unclear. The mechanism by which ANKRD49 establishes contact with JNK or p38 MAPK remains unknown. Accordingly, more in-depth research is needed to elucidate the function and underlying mechanisms of ANKRD49 in NSCLC progression.
We found that ANKRD49 potentiates the migration and invasion of H1299 and H1703 cells by activating the JNK-ATF2/c-Jun signaling cascade to upregulate MMP2 and MMP-9 expression.
All materials are available by contacting the corresponding author.
non-small cell lung cancer
lung squamous cell carcinoma
ankyrin repeat domain 49
short hairpin RNA
mitogen-activated protein kinase
c-Jun N-terminal kinase
activating transcription factor 2
cell counting kit-8
polymerase chain reaction
Real-time quantitative PCR
sodium dodecyl sulfate polyacrylamide gel electrophoresis
poly vinylidene fluoride membrane
nuclear export signal
nuclear localization sequence
Specific Pathogen Free
kelch like ECH associated protein 1
serine/threonine kinase 11
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This work was supported by funding from the Science and Technology Cooperation and Exchange Special Project of Shanxi Province (No. 202104041101012), the Natural Science Fund of Shanxi Province (No. 202201D111352), the Non-profit Central Research Institute Fund of Chinese Academy of Medical Science (No.2020-PT320-005).
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All tissue samples were obtained in accordance with the Declaration of Helsinki (1975) and were approved by the ethics committee of the First Hospital of Shanxi Medical University (approval number 2019K042). All procedures performed in studies involving human participants were performed per the ethical standards of the institution with written and verbal informed consent. All experiments in this study were performed in accordance with relevant guidelines and regulations and were approved by Ethics Committee of Shanxi Medical University. All animal experiments were approved by the Animal Ethics Committee of the Shanxi Medical University (approval number SYDL-2020016) and was performed according to the procedures that complied with the ARRIVE guidelines.
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Sun, J., Hu, Jr., Liu, Cf. et al. ANKRD49 promotes the metastasis of NSCLC via activating JNK-ATF2/c-Jun-MMP-2/9 axis. BMC Cancer 23, 1108 (2023). https://doi.org/10.1186/s12885-023-11612-9