Research ArticleExperimental Studies

14-3-3 Sigma Protein Contributes to Hepatocyte Growth Factor-mediated Cell Proliferation and Invasion via Matrix Metalloproteinase-1 Regulation in Human Gastric Cancer

JI YOON JUNG, SUNG AE KOH, KYUNG HEE LEE and JAE RYONG KIM
Anticancer Research January 2022, 42 (1) 519-530; DOI: https://doi.org/10.21873/anticanres.15510

Abstract

Background/Aim: The 14-3-3 protein family has a variety of functions in cellular responses in different organisms, including cell-cycle regulation, apoptosis, and malignant transformation. 14-3-3 Sigma protein (14-3-3σ) induces G2 arrest, which enables repair of damaged DNA. The purpose of this study was to identify the role of 14-3-3σ up-regulation by hepatocyte growth factor (HGF) in cancer cell proliferation and invasion in gastric cancer. Materials and Methods: In this study, cell culture, western blotting, real-time polymerase chain reaction, zymography, 14-3-3σ knock-down using short hairpin RNA (shRNA), electrophoresis mobility-shift assay, chromatin immunoprecipitation assay and standard two-chamber invasion assay were applied. Results: Firstly, we confirmed that the expression of 14-3-3σ in gastric cancer cells was up-regulated by HGF. To identify how HGF-induced 14-3-3σ expression affects matrix metalloproteinase-1 (MMP1) expression, the cells were treated with the mitogen-activated protein kinase kinase inhibitor PD098059 and analyzed using western blotting. The HGF-mediated expression of MMP1 protein decreased in the presence of PD098059. The role of 14-3-3σ in MMP1 expression was determined through 14-3-3σ knockdown using shRNA. 14-3-3σ-shRNA cells showed reduced levels of MMP1, phosphorylated extracellular signal-regulated kinase, and pp38. HGF-mediated cell proliferation and in vitro invasion were reduced in 14-3-3σ knockdown cells. Serum 14-3-3σ levels were also significantly reduced following gastrectomy in patients with stage II or stage III gastric cancer (p<0.05). Conclusion: These results suggest that 14-3-3σ plays an important role in cell proliferation and metastasis in gastric cancer, and 14-3-3σ may be a novel target for detection and prevention of progression of gastric cancer. In addition, the serum 14-3-3σ level is associated with treatment status in patients with locally advanced gastric cancer.

Key Words:

The HGF and MET proto-oncogene, receptor tyrosine kinase (MET) pathways play an important role not only in normal cell development but also in the pathogenesis of most types of human cancer (1-4). To explain the effects of HGF on the pathogenesis of gastric cancer, the gastric adenocarcinoma cell lines, NUGC3 and MKN-28, were screened using a human complementary DNA (cDNA) microarray in previous work, and many genes that are regulated by HGF were identified (5). That study suggested that calpain 12, cold-shock domain protein A, 14-3-3 proteins, cervical cancer 1 proto-oncogene, gastrin-releasing peptide, hepatoma-derived growth factor, mitogen-activated protein-binding protein-interacting protein and S100 calcium-binding protein A11 were up-regulated threefold or higher after treatment with HGF (2, 6).

The 14-3-3 proteins are a large family of small, acidic polypeptides of 28-33 kDa that are encoded by at least two different 14-3-3 genes in all eukaryotic species (7, 8). 14-3-3 Proteins are an important family for research because they have diverse cellular roles in signal transduction involved in cancer development, including cell -cycle regulation, apoptosis, and intracellular chaperones (9-12). In humans, the 14-3-3 protein family is composed of seven isoforms β, γ, ε, η, σ, τ (sometimes referred to as θ), and ζ; however, 14-3-3σ is the only isoform whose expression is restricted to epithelial cells (13, 14). The 14-3-3σ gene was discovered while screening for colorectal cancer cell genes that are differentially regulated during G2-M cell cycle arrest induced by DNA damage. This induction is dependent on TP53, and 14-3-3σ is induced by ectopic expression of p53 (15, 16). The p53 homologs such as p73 and p63 mediates the regulation of 14-3-3σ by the same binding motif (17). Furthermore, 14-3-3 proteins induce the apoptotic pathway in cultured cells in response to stimulation due to the activation of p38 mitogen-activated protein kinase (MAPK) (18, 19). 14-3-3σ released from keratinocytes was shown to induce the expression of matrix metalloproteinase-1 (MMP1), MMP8, and MMP24 in fibroblasts through the p38 MAPK signaling pathway (20-22). Some studies have reported that several MMP genes responsive to extracellular stimuli contain an activator protein-1 (AP-1) binding site in the proximal promoter in relation to the transcription initiation site. Another distal AP-1 or related element is found in the promoters of MMP1, MMP3, and MMP9. JUN and FOS bind to the AP-1 cis-element and activate the transcription of the MMP gene (23, 24). AP-1 complex is heterodimer composed of one Fos family member (FOSL1, FOSL2, FOSB, and c-FOS) and one JUN family member (JUNB, JUND, or c-JUN) (25-28). Furthermore, 14-3-3 proteins induced the apoptotic pathway in various cancer types, including colon and prostate cancer, in response to stimulation due to the activation of MAPK-related MMPs (15, 19, 29). These studies suggest that 14-3-3 proteins contribute to cancer development, especially the σ-isoform that is involved in cancer etiology through diverse signaling pathways.

In this study, we set out to determine the role of 14-3-3σ in gastric adenocarcinoma cell lines. We constructed small interference ribonucleic acid (siRNA) to use as tools for suppressing 14-3-3σ expression in the poorly-differentiated gastric adenocarcinoma cell line NUGC-3, and the moderately differentiated tubular gastric adenocarcinoma cell line MKN-28. In addition, we analyzed whether variation in serum 14-3-3 sigma levels in patients with stage II or stage III gastric cancer before and after surgery were statistically significant.

Materials and Methods

Blood samples. Blood samples were collected from patients with gastric cancer who underwent surgery including subtotal and total gastrectomy with or without lymph node dissection at Yeungnam University hospital between January 2010 and December 2012. A 10 ml blood sample was collected from each patient before and after surgery. This study was approved by the Institutional Review Board of the Yeungnam University Hospital (no. YUMC 2020-09-067). Written informed consent was obtained from each patient.

Cell culture. Two human gastric cancer cell lines (NUGC-3, MKN-28) were obtained from the Korea Cell Line Bank (Seoul, Republic of Korea). The cells were cultured in RPMI-1640 medium (Life Technologies Inc., Gaithersburg, MD, USA) containing 10% fetal bovine serum in an incubator under a humidified atmosphere of 5% CO2/95% air at 37°C.

Reagents and antibodies. Recombinant human HGF and human MMP1 antibody were purchased from R&D systems (R&D systems, Inc., Minneapolis, MN, USA). Horseradish peroxidase-conjugated anti-mouse and anti-rabbit antibodies were purchased from Bio-Rad Laboratories (Philadelphia, PA, USA). Anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were purchased from Cell Signaling Technology (Beverly, MA, USA). Anti-14-3-3σ, and rabbit polyclonal antibody against human JUNB, c-FOS and c-JUN were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). Mitogen-activated protein kinase (MAPK) kinase inhibitor PD098059 was purchased Biomol Research Laboratories, Inc. (Butler Pike, PA, USA). p38 inhibitor SB203580 was purchased from Calbiochem Inc. (San Diego, CA, USA).

14-3-3σ knockdown with short hairpin RNA (shRNA). The human 14-3-3σ-specific shRNA expression vector (14-3-3σ-shRNA) was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Following the instructions for Lipofectamine2000 (Invitrogen, Carlsbad, CA, USA), cells were transfected with 14-3-3σ-shRNA for 24 h and then selected for stable clones in medium containing puromycin (10 μg/ml) for another 2 weeks. Stable transfectant clones with low expression of the target genes were identified using western blot analysis.

3-(4, 5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cell proliferation was measured using the MTT assay. Control and 14-3-3σ shRNA-expressing cells (1.5×103 cells/well) were seeded in 96-well plates. After serum starvation for 24 h, control and 14-3-3σ shRNA-expressing cells were treated with or without HGF (10 ng/ml). After treatment for 72 h, 1 mg/ml MTT solution was added to the cells for 4 h. The formazan crystals were dissolved in DMSO (200 μl/well). Viability of each sample was assessed by measuring the absorbance at 570 nm using Bio-Rad multi-scan plate reader (Hercules, CA, USA).

Semiquantitative reverse transcription-polymerase chain reaction (RT-PCR). TRIzol (Invitrogen, USA) was used to isolate RNA from control cells treated with and without HGF (10 ng/ml). Complementary DNA (cDNA) was synthesized from total RNA using MMLV reverse transcriptase (Promega Corp., Madison, WI, USA) by the oligo (dT) priming method. PCR was performed in a 10 μl reaction volume containing 10 mM Tris-HCl pH 8.5, 50 mM KCl, 1 μl cDNA, 200 μM dNTPs, 1 mM MgSO4, 1 U platinum pfx Taq polymerase, and 2 μM primers. The primers used were as follows: 14-3-3σ; 5’-ttgtggctgagaactggaca-3’ (forward) and 5’-acaccca gcagacatgcttt-3’ (reverse), GAPDH; 5’-aggggtctacatggcaactg-3’ (forward) and 5’-cgaccactttgtcaagctca-3’ (reverse). The reaction cycle was as follows: 95°C for 4 min; followed by 30 cycles at 94°C for 15 s, 60°C for 15 s, and 72°C for 30 s and 72°C for 10 min. The PCR products were separated on a 1.5% agarose gel containing ethidium bromide and visualized on an ultraviolet transilluminator. SYBR Green (Roche applied Science, Indianapolis, IN, USA) real-time PCR was performed using a LightCycler1.5 (Roche Diagnostics, Almere, the Netherlands) according to the manufacturer’s suggestion. A dissociation curve was performed after each experiment to confirm a single product was amplified. A standard curve was generated for each gene using known copy numbers of a plasmid containing the cDNA specific to the gene.

Standard two-chamber invasion assay. Control and 14-3-3σ shRNA-expressing cells (1×104 cells) were seeded in the upper chamber of transwells (BD Biosciences, Bedford, MA, USA) with/without 10 ng/ml HGF in serum-free medium. After incubation for 48 h, the migrated cells on the lower membrane were fixed and stained using a HEMA 3 stain set (Curtis Matheson Scientific, Houston, TX, USA) according to the manufacturer’s instructions. The stained filter membrane was cut and placed on a glass slide. The migrated cells were counted under light microscopy (10 fields at 200× magnification).

Western blot analysis. Control and 14-3-3σ shRNA-expressing cells were harvested and extracted by RIPA lysis buffer with protease inhibitors (1 mM phenylmethanesulfonyl fluoride and 1 mM Na3VO4) and centrifuged at 18,928 × g, at 40°C for 10 min. The protein concentration of cell lysates was quantified by a BCA protein assay Kit (Thermo Scientific, Houston, TX, USA). The samples containing 50 μg of proteins were separated on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel and transferred to nitrocellulose membranes. The membranes were blocked with 5% skimmed milk in Twin-Tris-buffered saline (TTBS) for 30 min and then incubated overnight at 4°C with the following primary antibodies: anti-14-3-3σ (1:100), anti-JUNB (1:100), anti-c-FOS (1:100), anti-c-JUN (1:100) and anti-GAPDH (1:1,000). After washing six times with TTBS for 5 min, membranes were incubated with a horseradish peroxidase-conjugated secondary antibody (1:1,000 dilution) for 90 min at 4°C. The membranes were rinsed three times with TTBS for 30 min and antigen–antibody complexes were detected using the enhanced chemiluminescence detection system.

Zymography. NUGC-3 and MKN-28 cells were treated with HGF (0, 10, 40 ng/ml) for 48 h. The cell culture media were collected and were electrophoresed on a 10% polyacrylamide gel containing 0.1% (w/v) collagen I for MMP. The gel was incubated at room temperature for 2 h in the presence of 2.5% Triton X-100 and then incubated in developing buffer [10 mM CaCl2, 0.15 M NaCl, and 50 mM Tris (pH 7.5)] at 37°C overnight. After incubation, gels were stained with 0.25% Coomassie brilliant blue in methanol:acetic acid:water (4:1:5) solution and destained in the same solution, but without the dye. Collagenolytic activities were detected as the negatively-stained regions against a Coomassie blue-stained gel background. Zymographic analyses were performed in at least three independent experiments.

Electrophoresis mobility-shift assay. Nuclear extracts were prepared and electrophoresis mobility-shift assay was performed with electrophoresis mobility-shift assay kit (Pierce Biotechnology Inc., Rockford, IL, USA) according to the manufacturer’s instructions. A 10 μl reaction mixture containing 10 μg of nuclear protein was incubated for 30 min at room temperature with a binding mixture containing 10 mM Tris, 50 mM KCl, 1 mM dithiothreitol, 5 mM MgCl2, 2 μg poly (1 μg/μl in 10 mM Tris, 1 mM EDTA; pH7.5) and 2 pmol oligonucleotide probe, with or without a biotin-labelled AP-1 probe. Protein–DNA complexes were separated by electrophoresis on a 6% non-denaturing acrylamide gel containing 0.5 x TBE buffer (1x TBE- 90 mM Tris, 90 mM boric acid, 2.5 mM EDTA), transferred to Biodyne nylon membranes (Pierce), and then cross-linked in UV cross-linking for 10 min. For antibody super shift assay, 5 μg of anti-JUNB (Santa Cruz Biotechnology) was added to the reactions, followed by incubation for 45 min at room temperature before gel loading. Protein gel shifts were visualized using streptavidin-horseradish peroxidase followed by chemiluminescence detection.

Chromatin immunoprecipitation (CHIP) assay. The CHIP assay was performed using a CHIP assay kit (Upstate Biotechnology, Waltham, MA, USA) following the manufacturer’s directions. Briefly, control and 14-3-3σ shRNA-expressing cells adapted to serum-free media for 24 h were treated with/without 10 ng/ml HGF for 1 h. DNA and protein complexes were then cross-linked in situ with 1% formaldehyde. The cells were lysed, and chromatin was sheared into 200- to 1,000-b fragments by sonication. Antibodies recognizing AP-1 (1:100) were used for immunoprecipitation, and the chromatin fragments containing the crosslinked protein were purified by immune-absorption and elution from protein A/G beads. The crosslinks were reversed and the DNA was purified using a QIA quick nucleotide removal kit (QiAGEN Inc., Valencia, CA, USA). The DNA region crosslinked to the protein was determined by PCR analysis using the following primers designed for the MMP1 promoter (+19 to –159): 5’-caccaagtgattccaa-3’ (forward) and 5’-gctgctccaatatccc-3’ (reverse).

14-3-3σ Enzyme-linked immunosorbent assay (ELISA). The levels of abundance of 14-3-3σ in patient sera were measured using an ELISA kit (CUSABIO, Candler, NC, USA) according to the manufacturer’s instructions. Serum samples from patients with gastric cancer were measured and assayed simultaneously in duplicate. Serial dilutions of the 14-3-3σ standard were assayed in parallel with serum samples. The optical density was plotted against standard 14-3-3σ concentrations to generate a standard curve according to the manufacturer’s protocol. A non-parametric Wilcoxon rank-sum test was used to determine the significance of the difference between the median 14-3-3σ quantities in the sera before versus after surgery in patients with locally advanced gastric cancer. We tested the null hypothesis that data in the two observation groups are independent samples from identical continuous distributions with equal medians against the alternative hypothesis that the groups did not have equal medians. Significance was accepted at a two-tailed p-value less than 0.05.

Statistical analysis. Student’s t-test was used to analyze data. Results are expressed as the mean±standard deviation and p-values of less than 0.05 were considered statistically significant. The differences between pre-operative and post-operative 14-3-3σ levels were compared using a paired t-test.

Results

Up-regulation of 14-3-3σ after treatment with HGF. We used RT-PCR and western blotting to confirm that the expression of 14-3-3σ was up-regulated in gastric cancer cells treated with HGF. As expected, RT-PCR showed increased 14-3-3σ mRNA expression level following HGF treatment (Figure 1A). In addition, western blot analysis showed that 14-3-3σ protein level had increased 1 h after HGF treatment (Figure 1B). These results confirm that HGF treatment of gastric cancer cells increased mRNA and protein expression of 14-3-3σ.

Figure 1.
Figure 1.

Effects of hepatocyte growth factor (HGF) on the expression of 14-3-3σ in gastric cancer cells. Cells were serum-starved for 24 h, treated with 10 ng/ml HGF for the indicated times, and then harvested. The expression levels of 14-3-3σ RNA and protein were confirmed using reverse transcription-polymerase chain reaction (A) and western blot analysis (B). The figure illustrates representative data from three independent experiments. GAPDH: Glyceraldehyde 3-phosphate dehydrogenase.

Dose-dependent effects of HGF on 14-3-3σ and MMP1. Dose-dependent effects of HGF on 14-3-3σ were analyzed using western blotting. We found a concomitant increase in 14-3-3σ protein with increasing concentrations of HGF (0, 10, and 40 ng/ml) (Figure 2A). Western blot analysis and zymography were performed to validate MMP1 protein levels upon HGF treatment. The expression level of MMP1 protein increased in a dose-dependent manner with HGF treatment (Figure 2B). Zymography also showed that MMP1 activity increased with HGF treatment (Figure 2C). These results demonstrate that HGF affects tumor progression through up-regulation of 14-3-3σ and MMP1.

Figure 2.
Figure 2.

Effects of hepatocyte growth factor (HGF) on 14-3-3σ, matrix metalloproteinase-1 (MMP1) expression in gastric cancer cells. Cells were serum-starved for 24 h and treated with HGF at the indicated concentrations for 1 h then harvested. The expression levels of 14-3-3σ were confirmed using western blotting (A). The levels of MMP1 protein in the culture media were analyzed using western blotting (B) and zymography (C). The figure illustrates representative data from three independent experiments. GAPDH: Glyceraldehyde 3-phosphate dehydrogenase.

Effect of PD098059 and SB203580 on 14-3-3σ expression. It is well known that 14-3-3 proteins induce the apoptotic pathway in various cancer cells in response to stimulation by activating MAPK (15, 19, 29). Therefore, we examined whether MAPK and p38 are associated with HGF-induced 14-3-3σ. The cells were treated with either MAPK kinase inhibitor (PD098059) or p38 inhibitor (SB203580) and then analyzed via western blotting. The protein expression of 14-3-3σ induced by HGF was reduced in the presence of PD098059 and SB203580 in both NUGC-3 and MKN-28 cell lines (Figure 3).

Figure 3.
Figure 3.

Effects of mitogen-activated protein kinase (MAPK) kinase inhibitor PD098059 and of p38 inhibitor SB203580 on hepatocyte growth factor (HGF)-induced 14-3-3σ expression in gastric cancer cells. The cells (1×106/well) were plated overnight in complete medium, then starved for 24 h. Serum-starved cells were pretreated with 5, 10, and 20 μM of MAPK inhibitor (PD098059, PD) or p38 MAPK inhibitor (SB203580, SB) for 1 h and then treated with 10 ng/ml HGF. 14-3-3σ expression was evaluated using western blot analysis. The figure illustrates representative data from three independent experiments. GAPDH: Glyceraldehyde 3-phosphate dehydrogenase.

Effect of 14-3-3σ knockdown on HGF-mediated expression of MMP1. The effect of 14-3-3σ knockdown on MMP1 was measured to determine the relation of MMP1 and 14-3-3σ. We observed that the 14-3-3σ level decreased in both knockdown cell lines. Next, we examined the effect of 14-3-3σ knockdown on MMP1 with HGF treatment via western blotting. The level of HGF-mediated MMP1 expression decreased in both cell lines under 14-3-3σ knockdown. These results suggest that 14-3-3σ may regulate HGF-mediated MMP1 expression via the p38 and ERK signaling pathways (Figure 4).

Figure 4.
Figure 4.

Effects of 14-3-3σ knockdown using short hairpin RNA (shRNA) on hepatocyte growth factor (HGF)-induced expression of matrix metalloproteinase-1 (MMP1) in gastric cancer cells. Control and 14-3-3σ shRNA-expressing cells were starved for 24 h, and then treated with/without HGF (10 ng/ml) for 1 h and harvested. The expression levels of 14-3-3σ and MMP1 proteins in the culture media were analyzed using western blotting. The figure illustrates representative data from three independent experiments. GAPDH: Glyceraldehyde 3-phosphate dehydrogenase.

Effect of 14-3-3σ knockdown on HGF-mediated expression of c-FOS, c-JUN and JUNB. The effect of 14-3-3σ knockdown on AP-1 complex, which consists of c-FOS, c-JUN and JUNB, was measured to determine the relationship between AP-1 and 14-3-3σ. We examined the effect of 14-3-3σ knockdown with HGF treatment on JUNB expression using western blotting. The level of HGF-mediated JUNB expression was reduced in both cell lines under 14-3-3σ knockdown. In addition, we found that c-FOS and c-JUN decreased in both cell lines under 14-3-3σ knockdown (Figure 5). These results suggest that 14-3-3σ may regulate HGF-mediated MMP1 expression via AP-1 associated transcription.

Figure 5.
Figure 5.

Effects on JUNB, c-FOS and c-JUN of 14-3-3σ knockdown using short hairpin RNA (shRNA) in gastric cancer cells. Control and 14-3-3σ shRNA-expressing cells were starved for 24 h, and then treated with/without 10 ng/ml hepatocyte growth factor (HGF) for 1 h and harvested. The expression levels of JUNB, c-FOS and c-JUN were analyzed using western blotting. The figure illustrates representative data from three independent experiments. GAPDH: Glyceraldehyde 3-phosphate dehydrogenase.

Effect of 14-3-3σ knockdown on AP-1 binding to MMP1 promoter. We examined the putative binding sequence of AP-1 on the MMP1 promoter using a sequence analysis program to elucidate whether AP-1 regulates the transcriptional activity of MMP1 mRNA by binding to the MMP1 promoter (Figure 6A). To examine the function of AP-1 binding activity regulated by 14-3-3σ on the MMP1 promoter, we treated the 14-3-3σ knockdown and control cells with HGF and measured the binding activity of AP-1 to the putative binding site using CHIP assay. HGF treatment increased AP-1 binding to the MMP1 promoter but, in contrast, AP-1 binding was not observed in 14-3-3σ knockdown cells irrespective of HGF treatment (Figure 6B). These results suggest that 14-3-3σ may be associated with AP-1 binding to the MMP1 promoter.

Figure 6.
Figure 6.

Effect on matrix metalloproteinase-1 (MMP1) promoter of hepatocyte growth factor (HGF)-mediated regulation of 14-3-3σ. Control and cells with 14-3-3σ knockdown using short hairpin RNA (shRNA) were starved for 24 h, and then treated with/without HGF (10 ng/ml) for 1 h and harvested. Chromatin immunoprecipitation assay results show the amplification of a fragment of the proximal MMP1 promotor containing activator protein-1 binding site (A). Immunoprecipitation was carried out using an antibody to 14-3-3σ (B).

Determination of binding activity of AP-1 under 14-3-3σ knockdown in HGF-treated cells using electrophoresis mobility shift assay. We treated 14-3-3σ knockdown and control cells with HGF and measured the binding activity of AP-1 using electrophoresis mobility shift assay to examine the function of AP-1 binding activity as regulated by 14-3-3σ. The results showed that AP-1 binding activity increased in HGF-treated cell lines, especially that of JUNB. In contrast, AP-1 binding activity decreased in both 14-3-3σ shRNA gastric cancer cell lines (Figure 7).

Figure 7.
Figure 7.

Effect of hepatocyte growth factor (HGF) on 14-3-3σ-induced JUNB and AP-1 activation. Cells were serum-starved for 24 h, treated with HGF for 1 h and then harvested. The expression levels of JUNB were confirmed using western blotting (A). Control and cells with 14-3-3σ knockdown using short hairpin RNA (shRNA) were treated with 10 ng/ml HGF for 1 h and nuclear extracts were prepared. The specific DNA binding activity of AP-1 transcription factor was determined by electrophoresis mobility shift assay in the presence or absence of nuclear extracts. Anti-JUNB was employed for super shift analysis (B).

Effect of 14-3-3σ knockdown on HGF-mediated proliferation. The 14-3-3σ shRNA cells and control cells were treated without or with HGF and cell proliferation was measured after 72 h using MTT assay. The results showed that HGF-mediated proliferation was significantly lower under 14-3-3σ knockdown in both gastric cancer cell lines (p<0.05) (Figure 8).

Figure 8.
Figure 8.

Effects of 14-3-3σ knockdown using short hairpin RNA (shRNA) on cell proliferation in gastric cancer cells. Control and 14-3-3σ shRNA cells (1.5×103 cells) were seeded in 96-well plates. After serum starvation for 24 h, cells were treated with/without 10 ng/ml of hepatocyte growth factor (HGF) for 72 h. Cell proliferation was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays and expressed as a ratio to that of untreated non-transfected control cells. Values are presented as the mean±standard deviation of experiments performed in triplicate.

Effect of 14-3-3σ knockdown on HGF-mediated cell invasion. To identify whether 14-3-3σ plays a role in cell invasion, we performed an in vitro invasion assay using Matrigel-coated migration chambers. We treated 14-3-3σ shRNA cells and control cells with HGF and used them for the assay. After 72 h, we found that HGF-mediated cell invasion decreased under 14-3-3σ knockdown in both gastric cancer cell lines (p<0.05) (Figure 9).

Figure 9.
Figure 9.

Effect of 14-3-3σ knockdown using short hairpin RNA (shRNA) on hepatocyte growth factor (HGF)-mediated invasion gastric cancer cells. Control and 14-3-3σ shRNA cells were starved for 24 h, and then treated with/without HGF (10 ng/ml) for 48 h. Cell invasion capacity was measured using a standard two-chamber invasion assay with Matrigel migration chambers and expressed as a ratio to that of untreated non-transfected control cells. Values are presented as the mean±standard deviation of three independent experiments.

Analysis of 14-3-3σ levels in serum from patients with gastric cancer. Following all these assays, it could be postulated that 14-3-3σ mediates cancer cell invasion and metastasis associated with MMP1 via the MAPK, p38 and AP-1 pathways. We hypothesized that 14-3-3σ levels in patients’ blood would be lower after surgery than before. To test this, we obtained blood samples from 37 patients with gastric cancer before and after gastric cancer surgery, and estimated the 14-3-3σ levels using ELISA

Of these patients, 25 (67.6%) were male and 12 (32.4%) were female. The median age was 61.3 years (range=39-76 years), and 17 patients (45.9%) had stage I disease. The mean preoperative 14-3-3σ level in these patients was 0.093 ng/ml while the postoperative 14-3-3σ level was 0.08668 ng/ml (p=0.254). A statistically significant reduction in 14-3-3σ levels was observed between samples collected before and after surgery in patients with stage II and stage III disease (p<0.05) (Figure 10). In addition, when the mean difference in 14-3-3σ levels in samples were stratified based on patient characteristics, namely sex, tumor size, stage, Lauren classification, lymph node metastasis and lymphatic, neural and serosal invasion, no statistically significant differences were observed (Table I).

Figure 10.
Figure 10.

Serum from 37 patients prior to and after surgery for stage I (A) or stage II/III (B) gastric cancer was evaluated for 14-3-3σ quantity using enzyme-linked immunosorbent assay. In patients with stage I disease, there was no statistically significant difference in the preoperative and postoperative 14-3-3σ levels but in patients with stage II or III disease, the postoperative 14-3-3σ level was significantly reduced (*p<0.05).

View this table:
Table I.

Mean difference (post-preoperative) in serum levels of 14-3-3σ in 37 patients with gastric cancer according to clinicopathological features.

Discussion

Gastric cancer is the most common malignant tumor, not only in Northeast Asia but also worldwide, and is the second most common cause of cancer-related deaths (30, 31). It is characterized by rapid progression and metastasis to other organs, late clinical presentation, and poor survival rates. Although, many anticancer drugs have been developed and others are in the development pipeline, such as targeted agents and immune-oncological drugs, gastric cancer is still difficult to treat. There is, therefore, an urgent need for the identification of novel targets that can be exploited to suppress or prevent progression and metastasis. Tumor progression and metastasis must go through a multistep process to spread from the primary tumor, including migrating through the extracellular matrix (32-34). MMPs are a family of zinc-containing proteases (35) involved in the degradation and migration of different components of the extracellular matrix, and there is evidence suggesting that individual MMPs play important roles in tumor progression and metastasis (36, 37). Studies have reported that MMPs and their levels are associated with tumor grade and invasion in various human cancer types (29, 38, 39). A large proportion (94%) of gastric tumors were found to contain MMP2, and MMP1, and MMP9 were found in in 73% and 70%, respectively (40). Another study reported that 14-3-3σ is associated with cell surface aminopeptidase N in the regulation of MMP1 and 14-3-3σ released from keratinocytes induced the expression of MMPs in fibroblasts through the p38 MAPK signaling pathway (41).

The 14-3-3 proteins are encoded by at least two different genes in all eukaryotic species (7, 8), and the 14-3-3 proteins are an important family for research because they have diverse cellular roles, including cell-cycle regulation and apoptosis, as well as being intracellular chaperones in signal transduction in cancer development (9-12). In humans, the 14-3-3 protein family is composed of seven isoforms; however, 14-3-3σ is the only isoform that is expressed exclusively in epithelial cells (13). According to other studies, 14-3-3 proteins are associated with cancer, and 14-3-3σ, in particular, has a direct cancer connection (7, 14).

The present study distinctly showed 14-3-3σ to be up-regulated in NUGC-3 and MKN-28 cells treated with HGF (Figure 1) and that HGF-mediated up-regulation of 14-3-3σ is involved in the regulation of MMP1 via the p38 MAPK and AP-1 pathways (Figure 2, Figure 4 and Figure 5). In addition, 14-3-3σ was shown to exert its effects by binding to the MMP1 promoter (Figure 6 and Figure 7), and cell invasion and proliferation decreased in 14-3-3σ knockdown cell (Figure 8 and Figure 9).

Based on these findings, it was hypothesized that the level of 14-3-3σ would i) decrease after the removal of gastric tumor and lymph node metastases during gastric cancer surgery; and ii) be higher before surgery in patients with advanced gastric cancer compared to those with early gastric cancer. Analysis of serum 14-3-3σ in 37 patients who underwent gastric cancer surgery showed a statistically significant reduction in the mean 14-3-3σ level after surgery in patients with stage II and III gastric cancer (p<0.01) (Figure 10B). In patients with stage I, there was no statistically significant difference between the preoperative and postoperative 14-3-3σ levels (Figure 10A), but in stage II and III, the postoperative 14-3-3σ levels were significantly reduced (Figure 10B). Therefore, the results of the present study showed that patients with locally advanced gastric cancer have higher levels of 14-3-3σ before surgery and 14-3-3σ might affect the progression and metastasis of gastric cancer. This is the first study in which quantitative analysis of 14-3-3σ was performed using ELISA, and it provided evidence that clinical data supported the findings of cell experiments. However, 14-3-3σ expression in serum may not always be induced by HGF, and statistical significance cannot be generalized due to the small number of samples in this study. Therefore, proving a relationship between HGF and 14-3-3σ will require the analysis of a larger number of samples before 14-3-3σ can be considered a promising target for the detection of proliferation and metastasis in gastric cancer.

In conclusion, the results of the present study indicate that 14-3-3σ plays an important role in up-regulation of MMP1 and contributes to HGF-mediated cancer progression and metastasis. 14-3-3σ may be an important factor in the biology of tumor progression and metastasis, and may be a promising target for gastric cancer treatment.

Acknowledgements

The Authors gratefully acknowledge Innovation Center for Aging Research (i-CARE), College of Medicine, Yeungnam University for the supports this study.

Footnotes

  • Authors’ Contributions

    Conception and design: K.H. Lee. Development of methodology: J.Y. Jung and K.H. Lee. Acquisition of data: J.Y. Jung, S.A.Koh and K.H. Lee. Analysis and interpretation of data: J.Y. Jung and K.H. Lee. Writing, review, and/or revision of the article: J.Y. Jung. Administrative, technical, or material support: J.Y. Jung, S.A.Koh and K.H. Lee. Study supervision: J.R.Kim. All Authors read and agreed to the published version of the article.

  • Conflicts of Interest

    The Authors have no conflicts of interest to declare.

  • Received August 17, 2021.
  • Revision received November 9, 2021.
  • Accepted November 16, 2021.

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14-3-3 Sigma Protein Contributes to Hepatocyte Growth Factor-mediated Cell Proliferation and Invasion via Matrix Metalloproteinase-1 Regulation in Human Gastric Cancer
JI YOON JUNG, SUNG AE KOH, KYUNG HEE LEE, JAE RYONG KIM
Anticancer Research Jan 2022, 42 (1) 519-530; DOI: 10.21873/anticanres.15510
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