GATA6 inhibits the biological function of non-small cell lung cancer by modulating glucose metabolism

Tissue specimens of patients with NSCLC undergoing surgical resection in Dandong First Hospital from January 2010 to December 2013 were collected. The inclusion criteria were as follows: (1) None of the patients had received radiotherapy, chemotherapy or other treatment before surgery; (2) The tissue specimen was confirmed as lung adenocarcinoma by histopathology. A total of 150 patients were collected, ranging in age from 38 to 86 years, with a median age of 60 years. A total of 101 cases were followed up with complete information. This study was approved by the Ethics Committee of Dandong First Hospital, and all patients signed informed consent.

Antibodies GATA6 (1:200, ab175349, abcam), c-Myc (1:200, ab32072, abcam), Ki67 (1:1000, ab15580, abcam) and Cleaved Caspase-3 (1:200, 9961, CST) were used. The first antibody diluent for immunohistochemistry, the second antibody labeled with horseradish peroxidase (PV-6000) and the DAB color development kit were all purchased from Beijing Zhongshan Jinqiao Biotechnology Co., LTD. SP two-step method was used for staining. The primary antibodies were incubated at 4 °C overnight, and the secondary antibody was incubated at room temperature for 1 h, then the color was developed by DAB for 2 min. For the immunohistochemical results of GATA6, the number of positive cells and positive intensity were comprehensively considered, and a semi-quantitative method was adopted to score 0 points (0), 1 points (1–25%), 2 points (26–50%), 3 points (51–75%), and 4 points (76–100%) according to the proportion of positive cells, and 0 (no staining), 1 (lower staining), 2 (moderate staining), and 3 (higher staining) according to the coloring intensity. The two scores were multiplied, and 10 high-power field scores were randomly selected for each section in the hot spot area. Finally, all cases were divided into two groups according to the score results, 0–4 was divided into low-expression group and 5–12 was high-expression group. Immunohistochemical results of c-Myc, Ki67 and Cleaved Caspase-3 were analyzed statistically according to the percentage of positive cells.

Human lung adenocarcinoma cell lines A549 and PC9 were obtained from the ATCC, and cultured in medium 1640 containing 10% fetal bovine serum at 37 °C in a constant temperature incubator with 5% CO₂ volume fraction and saturated humidity. We confirmed the authenticity of all cell lines through short tandem repeat profiling and conducted tests to ensure the absence of Mycoplasma contamination. The plasmids and lentiviruses with overexpression or down-expression of GATA6, as well as the control plasmids and viruses were synthesized and packaged by Gene-Chem (Shanghai Genechem Co., Ltd.). The lung cancer cells were planted on the six-well plate, and the amount of virus required was calculated according to the number of infections to infect the cells of logarithmic growth stage. After 48 h, cells were screened with 1 mg/L purinamycin for 2 days. Then, the expression level of GATA6 was detected by real-time PCR and western blot to judge the infection effect.

Total cell RNA was extracted using TRIzol reagents (Invitrogen), quantified using NanoDrop 1000 (Thermo Fisher), and its quality was evaluated by gel electrophoresis. Real-time PCR was performed using Power SYBR Green (TaKaRa Company) with GAPDH as the internal control. The primers were: GATA6 upstream primer 5′-TGCAATGCTTGTGGACTCTA-3′, downstream primer 5′-GTGGGGGAAGTATTTTTGCT-3′; GAPDH upstream primer 5′-AAGGTGAAGGTCGGAGTCAA-3′, downstream primer 5′-AATGAAGGGGTCATTGATGG-3′.

Protein was extracted from RIPA lysate (Solebao), and protein concentration was measured and homogenized by BCA method. 10% SDS-PAGE gel electrophoresis was performed for 90 min, and the protein was wet-transferred on PVDF membrane by Bio-Rad device for 250 mA for 2 h. The PVDF film containing the target strip was cut and sealed with 5% milk for 1 h at room temperature. GATA6 antibody (1:1000), c-myc antibody (1:1000), GAPDH (Santa Cruz Corporation, 1:5000) and β-actin (Santa Cruz Corporation, 1:5000) were incubated overnight at 4 °C. Incubation of the secondary antibody (Cell Signaling Technology, 1:10,000) at room temperature for 1 h and ECL development (millipore).The Image J software was employed for the quantification of protein band intensities, with the ratio between the gray value of the target protein band and that of the internal reference protein band serving as a metric for assessing the relative expression of the target protein.

The cells with logarithmic growth were inoculated into 96-well plates (5 × 10³ cells /200 μL) with five replicates in each group. At 24-, 48-, 72-, 96-, and 120-h time points, respectively, the original culture medium was discarded and 100 μL culture medium containing 10% CCK-8 was added. After incubation for 2 h, OD values of each hole were measured at 450 nm, and growth curves were drawn.

Cells in the logarithmic growth phase were seeded into six-well plates at a density of 1000 cells per well and incubated for a period ranging from 10 to 14 days. Upon the emergence of visible colonies, the supernatant was aspirated, and the clones were fixed by adding methanol at −20 °C for 6 min. Subsequently, a 0.1% crystal violet solution was applied at room temperature for 30 min, and the number of colonies containing more than 50 cells was enumerated using an optical microscope.

The cell lines at logarithmic growth stage were taken for the preparation of single-cell suspension. After centrifugation, 10,000 cells were added to the upper chamber of the cell, and 500 μl of 10% FBS was added to the 24-well plate, and the cell was placed in the 24-well plate. After 24 h of culture, the cells in the upper chamber that did not cross the compartment membrane were wiped with cotton swabs. After crystal violet staining, the images were collected under the microscope. Dozens of visual fields were randomly counted under a 40-fold optical microscope for statistical analysis.

Using the Promega Dual-Luciferase™ Reporter Gene (DLR™) assay system according to the instructions, the c-Myc reporter plasmid and control plasmid were constructed by Gene-Chem (Shanghai Genechem Co., Ltd). Cells were inoculated into 96-well plates at a density of 2 × 10³ cells per well, and corresponding plasmids were transfected. After 24 h, the cells were lysed and the activity of firefly and kidney cells was measured. The luciferase signal was normalized with renilla values.

The expression level of GATA6 mRNA in lung adenocarcinoma and normal lung tissues was analyzed by interactive gene expression profile analysis of TCGA database (GEPIA, http://​gepia.​cancer-pku.​cn/​). Using KMPLOT database (http://​kmplot.​com/​analysis/​) analysis GATA6 mRNA on the survival of patients with lung adenocarcinoma.

All experimental data were subjected to statistical analysis using SPSS 16.0 software. The χ² test was employed to assess the correlation between the expression level of GATA6 protein and the clinicopathological indicators of the patients. Kaplan–Meier univariate survival analysis was conducted to investigate the association between the expression level of GATA6 protein and the patients' survival prognosis. In cell experiments, a t-test was utilized for statistical analysis to compare differences between the experimental and control groups. A significance level of P < 0.05 was considered statistically significant in all analyses. Each experiment was independently repeated more than three times to ensure robustness and reliability of the results.

To delineate the expression pattern of GATA6, we initially conducted an analysis of GATA6 mRNA expression levels in lung adenocarcinoma and normal lung tissues, utilizing data sourced from the TCGA database. Our findings unequivocally demonstrated that GATA6 mRNA expression in lung adenocarcinoma tissues was markedly diminished compared to that in normal lung tissues (Fig. 1A). Subsequently, we procured 10 pairs of fresh NSCLC tissues, along with their corresponding adjacent normal lung tissues, revealing a consistent reduction in both mRNA levels (Fig. 1B) and protein levels (Fig. 1C) of GATA6 within NSCLC specimens relative to normal lung tissues. Furthermore, immunohistochemistry (IHC) analyses performed on 150 patients corroborated these observations, manifesting a pronounced decrease in GATA6 expression in NSCLC tissues (Fig. 1D). Based on these IHC results, we categorized the 150 cases into two groups: the GATA6 low-expression group and the GATA6 high-expression group (Fig. 1E). Intriguingly, as shown in Table 1, we established a significant negative correlation between GATA6 expression levels and crucial clinicopathological parameters, including tumor volume (Fig. 1F), lymph node metastasis (Fig. 1G), and clinical staging of patients (Fig. 1H). Notably, patients exhibiting low GATA6 expression levels in NSCLC tissues displayed a considerably poorer overall survival (Fig. 1I) and disease-free survival (Fig. 1J). Furthermore, our multivariate survival analysis showed that GATA6 was an independent prognostic factor in patients with NSCLC (n = 101, Table 2). Additionally, statistical analyses conducted using the KMPLOT database corroborated the prognostic significance of GATA6 mRNA expression in lung cancer patients (Fig. 1K, L). Meanwhile, we harnessed data from the TCGA and KMPLOT databases to scrutinize the expression profiles of other GATA family members in lung adenocarcinoma tissues (Fig. 2A) and their associations with the prognosis of lung cancer patients (Fig. 2B). Among the six family members, GATA6 exhibited the strongest correlation with the prognosis of lung cancer patients, implying a more pronounced involvement of GATA6 in the context of lung cancer.

To ascertain the functional role of GATA6 in lung cancer cells, we generated cell lines with GATA6 overexpression (Fig. 3A, B), and our findings revealed a substantial inhibition of both proliferative capacity (Fig. 3C) and clonogenic potential (Fig. 3D). Moreover, the overexpression of GATA6 effectively restrained the migratory capabilities of lung cancer cells (Fig. 3E). Conversely, when GATA6 was silenced (Fig. 3F, G), it resulted in increased proliferation (Fig. 3H), heightened clonogenicity (Fig. 3I), and enhanced migration abilities (Fig. 3J) in A549 and PC9 cells. These results collectively underscore the suppressive role of GATA6 in regulating the malignant biological behavior of lung cancer cells.

During the cell culture process, noteworthy alterations were observed when comparing cells overexpressing GATA6 to control cells. Specifically, the medium pH of GATA6-overexpressing cells was found to be elevated (Fig. 4A), while the level of lactic acid within the medium was reduced (Fig. 4B). Conversely, GATA6-expressing cells exhibited an increase in the secretion of lactate (Fig. 4C). Additionally, GATA6 expression levels were found to be intricately linked to glucose absorption and utilization. The overexpression of GATA6 hindered the uptake and utilization of glucose by lung cancer cells (Fig. 4D), whereas decreased GATA6 expression promoted glucose uptake and utilization (Fig. 4E). Given the alterations in the metabolic phenotype of lung cancer cells, we sought to determine whether GATA6 expression levels influenced the expression of glucose-related metabolic enzymes. The results revealed that GATA6 overexpression suppressed the expression of several glucose-metabolizing enzymes as well as the glucose transporter GLUT1 (Fig. 4F, H), whereas decreased GATA6 expression fostered the expression of these proteins (Fig. 4G, I). Consequently, isotope experiments illustrated that GATA6 overexpression decelerated the metabolic flux of glucose (Fig. 4J). These findings collectively indicate that GATA6 modulates the metabolic phenotype of lung cancer cells by exerting influence over the expression of glucose-related metabolic proteins. Furthermore, the addition of 2-DG to cells with reduced GATA6 expression inhibited glucose metabolism and partially mitigated the increase in cell proliferation induced by decreased GATA6 expression (Fig. 5A). Moreover, GATA6 exhibited no significant impact on lung cancer cell proliferation in a sugar-free medium (Fig. 5B,C), underscoring its primary role in regulating glucose metabolism.

C-Myc is a major transcription factor that regulates the expression of metabolic enzymes related to glucose metabolism (Fatma et al. 2022; Duffy et al. 2021; Panda et al. 2020). We aimed to investigate whether GATA6 could impact the expression of relevant metabolic enzymes by modulating c-Myc expression. Initially, our findings revealed that GATA6 overexpression suppressed the expression of c-Myc mRNA in lung cancer cells (Fig. 6A, B), whereas GATA6 underexpression produced the opposite effect (Fig. 6C, D). Furthermore, a negative correlation between GATA6 mRNA and C-MYC mRNA levels was discerned in lung cancer tissue samples (Fig. 6E). The results of IHC also corroborated these findings, indicating lower c-Myc protein expression in lung cancer tissues exhibiting higher GATA6 levels (Fig. 6F). Subsequent luciferase reporter assays provided evidence that GATA6 indeed regulated c-Myc expression (Fig. 6G, H). Moreover, the utilization of a c-Myc inhibitor mitigated the proliferation of lung cancer cells induced by GATA6 silencing (Fig. 6I), concurrently inhibiting the upregulation of mRNA expression of genes associated with glucose metabolism induced by GATA6 silencing (Fig. 6J, k). These results collectively underscore that GATA6 modulates the expression of genes implicated in glucose metabolism by regulating c-Myc expression, thereby exerting regulatory control over the functionality of lung cancer cells.