CANT1 serves as a potential prognostic factor for lung adenocarcinoma and promotes cell proliferation and invasion in vitro

The lung adenocarcinoma tissues and adjacent tissues included in our study were from the TCGA database, where a total of 502 LA patients from 1991 to 2013. Gene expression data and the corresponding clinical information of the TCGA-LA were obtained from the TCGA (https://​portal.​gdc.​cancer.​gov/​). The GEO databases of GPL570 and GPL6884 platforms were performed for the validation (www.​ncbi.​nlm.​nih.​gov/​geo). Expression profiles (HTSeq-Counts) were compared between high and low CANT1 expression groups to identify differentially expressed genes (DEGs) using the DESeq2 R package. The data used in this study were in accordance with the guidelines provided by TCGA and GEO. The ethics approval and informed consent were not required.

We explored the expression differences of CANT1 in LA patients between tumor tissue and normal tissue. Besides, the immunohistochemistry staining of CANT1 expression was obtained by the Human Protein Atlas (HPA) (https://​www.​proteinatlas.​org) [19, 20]. On the HPA website, antibodies HPA019627, HPA022818, and HPA019639 were used for immunohistochemistry staining of CANT1.

A total of 502 LA patients were included in our study. The survival curve was performed by survival R package and survminer R package. Furthermore, we validated the prognostic values of the risk model in two independent datasets from GSE31219 (n = 83) and GSE41271 (n = 181).

A nomogram was built based on the multivariate analysis’s result to predict the survival probability for 1-year, 3-years, and 5-years of LA patients. The rms R package was performed to produce a nomogram. The concordance index (C-index) and calibration curve were performed by the Hmisc R package. In this study, C-index was carried out to determine the nomogram’s discrimination with 1000 bootstrap replicated.

GEPIA is a database based on the UCSC Xena project [21]. In the study, GEPIA 2.0 (http://​gepia2.​cancer-pku.​cn/​) was performed to find the gene with the most similar expression of CANT1 in LA. Finally, 50 most correlated co-expression genes were founded. Then the functional analysis of CANT1 and its co-expression genes in TCGA-LA were predicted by Metascape (http://​metascape.​org) [22].

Gene set enrichment analysis (GSEA) is a calculation method that determines whether a set of prior defined genes show statistically significant and consistent differences between two biological states [23, 24]. In this study, GSEA was performed using the Molecular Signatures Database (MSigDB) Collection (c2.all.v7.0.entrez.gmt) of clusterProfiler R package to illuminate the statistically significant pathway difference between high and low CANT1 expression groups of LA. The expression level of CANT1 was used as a phenotype label. The pathway terms with adjusted P-value < 0.05 and false discovery rate (FDR) q-value < 0.25 were considered significantly enriched.

The previous GSEA analysis suggested that CANT1 was related to DNA methylation. Therefore, we then analyzed the relationship between the expression of CANT1 and DNA methylation. Spearman correlation coefficient was performed to reveal the correlation between the expression of CANT1 and methylation level in TCGA-LA. The gene methylation data of the TCGA-LA project was obtained from the UCSC Xena browser (version:07-20-2019, https://​xenabrowser.​net/​datapages/​). The relationship between the methylation level and overall survival (OS) of CANT1 was retrieved from MethSurv (https://​biit.​cs.​ut.​ee/​methsurv/​). It is a web tool to provide univariable and multivariable survival analysis based on DNA methylation biomarkers by using TCGA data [25].

Human lung adenocarcinoma cell lines A549 and H1299 were obtained from the Genechem company (Shanghai, China). A549 cells were cultured in Ham’s F-12 K medium (PYG0036, Boster, USA) supplemented with 10% fetal bovine serum (FBS, P30-3302, PAN biotech, Germany) and 1% penicillin/streptomycin (100x, Gibco, USA), H1299 Cells were cultured in RPMI-1640 (PYG0006, Boster, USA) supplemented with 10% FBS and 1% penicillin/streptomycin. All cell lines were maintained in a humidified incubator with 5% CO2 at 37 °C. The medium was changed every 3 days, and a subcultivation ratio of 1:3 was performed.

Small interfering RNAs (siRNAs) were used to selectively knockdown the expression of CANT1 by the Lipofectamine 3000 transfection reagent (L3000-015, Invitrogen, USA), according to the manufacturer’s instructions. The sequence of three groups of siRNAs targeting CANT1 (siRNA-987, siRNA-1173, and siRNA-273) and two negative control groups (si-NC and NC) were obtained from the zolgene (Fuzhou, China) shown in Table 1. For transfection, A549 and H1299 cells in the logarithmic growth phase were inoculated into a six-well plate. Knockdown efficiency was verified on the RNA level using quantitative real-time polymerase chain reaction (qRT-PCR).

Total RNA was isolated using the TRIzol™ Reagent (15,596,026, Invitrogen, USA). Then the GoScript™ Reverse Transcription System (A5001, Promega, USA) was employed to reversely transcribe RNA into cDNA. qRT-PCR was conducted using UltraSYBR Mixture (CW0957, Cwbio, China) to measure the expression of CANT1. β-actin was used as the internal reference. The calculation of inhibitory efficacy of the CANT1 mRNA was calculated using the 2^-ΔΔCt method. The sequences of primers used in qRT-PCR were shown in Table 2.

After transfection, we used the CCK-8 reagent (40203ES60, Yeasen, Shanghai, China) to measure cell viability. The si-CANT1 group, si-NC group, and NC group of A549 and H1299 cells were subcultured in a 96-well plate and then incubated for 0, 24, 48, and 72 h. Before detection, 10 μl (10%) CCK8 reagent was added daily to each hole in the 96-well plates and incubated at 37 °C for 1 h. Then, a microplate reader (Nano-100, Allsheng, Hangzhou, China) was used to measure the absorbance at 450 nm.

The invasive ability of the A549 and H1299 cells was assessed with Transwell chambers and Matrigel. The Transwell chambers were coated with 50 μl diluted Matrigel (1:8) at 37 °C for 4 h. Subsequently, 1 × 10⁵/mL cells resuspended in serum-free medium were added into the Transwell chamber, which was placed in cell culture medium supplemented with 10% FBS and was cultured for 48 h. Then the residual cells in the upper chamber were wiped with cotton swabs. Cells migrated through the membrane were fixed with 4% paraformaldehyde (P1110, Solarbio, Beijing, China), washed with PBS, mixed with 0.4% crystal violet solution (C0121, Beyotime, Shanghai, China), and counting in 3 randomly picked view under a microscope at 100× magnification (MF53, Mshot, Guangzhou, China).

For cell cycle detection, 1 × 10⁶/ml cells were counted and washed with phosphate buffer saline (PBS, ABA212278, Hyclone, USA), followed by the addition of 70% cold ethanol at 4 °C overnight. After washing with cold PBS, the fixed-cell pellets were collected by centrifugation and resuspended in ribonuclease (RNase) A/Propidium Iodide (PI) staining buffer. The cell-cycle distribution was analyzed by flow cytometer (FACSVerse, BD, USA).

All statistical analysis was performed using SPSS (version 26.0) and R (version 4.0.2). Wilcoxon rank-sum test and Wilcoxon signed-rank test were used to compare the expression of CANT1 between LA and normal groups. X-tile software (version 3.6.1, https://​medicine.​yale.​edu/​lab/​rimm/​research/​software/​) was performed to seek the optimal cutoff value of CANT1 expression. The relationship between the TN stage and the expression of CANT1 was analyzed with Wilcoxon single-rank test. Clinical parameters (age, gender, number pack years smoked, epithelial growth factor receptor (EGFR) mutation status, kirsten rat sarcoma viral oncogene (KRAS) mutation status, anaplastic lymphoma kinase (ALK) mutation status, T stage, N stage, M stage, pathological stage, and gene expression) associated with OS in LA patients were evaluated with Cox analyses. In univariate analysis, all factors with P < 0.20 were involved in multivariate analysis to identify independent prognostic factors. Owing to missing data exceeding 20%, the ALK mutation status and M stage were not included in multivariate analysis. Comparisons between two groups were analyzed by t-test. All experiments were performed with at least three independent replications. All tests were two-sided, and a P-value < 0.05 was considered statistically significant.

The clinical data including 502 patients were collected from TCGA (Table 3).

We used the Wilcoxon rank-sum test to investigate the expression of CANT1 in 502 LA tissues and 57 normal tissues. The expression of CANT1 was significantly higher in LA groups than in normal groups (P < 0.001) (Fig. 1A). In addition, we further used Wilcoxon singed-rank test to examine the expression of CANT1 in 57 pairs of LA tissues. The result showed CANT1 was significantly overexpressed in LA (P < 0.001) (Fig. 1B). As shown in Fig. 1C-D, increased CANT1 expression in LA was significantly associated with the TN stage (T1 vs. T2/T3/T4, P = 0.042; NO/ N1 vs. N2/N3, P = 0.041). We also carried out an analysis of the association between CANT1 expression and M (metastasis) status. Due to the result are negative and meaningless, we did not add this result to the Figure. These results demonstrated that the CANT1 expression level could affect LA patients’ prognosis with different TN stage. After exploring the CANT1 expression patterns of CANT1 in LA, we tried to examine the protein expression patterns of CANT1 in LA by the HPA. As shown in Fig. 1E-J, low protein expressions of CANT1 were observed in normal lung tissues, while high protein expressions were detected in LA tissues. The staining intensity was scored as negative, weak and negative in HPA019627, HPA022818, and HPA019639 in normal lung tissues. While the staining intensity was scored as Strong in HPA019627, HPA022818, and HPA019639 in LA tissues. The quantity was scored as none, < 25%, and none in HPA019627, HPA022818, and HPA019639 in normal lung tissues. While the quantity was scored as > 75, > 75, and 25%-75% in HPA019627, HPA022818, and HPA019639 in LA tissues.

Overall survival (OS) was significantly reduced in patients with high CANT1 expression than those with low CANT1 expression (P = 0.005) (Fig. 2A). To validate the association between the expression of CANT1 and OS, we also performed the GSE30219 and GSE41271 datasets from the GEO. The outcome indicated that OS was significantly reduced in patients with high CANT1 expression than those with low CANT1 expression (P = 0.018, P = 0.043, respectively) based on GSE30219 and GSE41271 database (Fig. 2B-C). We further used the univariate Cox regression model to identify prognostic factors in LA (Table 4). The result showed that high CANT1 expression levels were correlated with worse OS (P = 0.005, hazard ratio [HR] = 1.547 (95% confidence interval [CI] [1.143-2.094])). In addition, age > 70 years old (P = 0.013, HR = 1.464(95% CI [1.083-1.979]), higher TNM stage (T: P = 0.004, HR = 1.666 (95%CI [1.182-2.347]); N: P < 0.001, HR = 2.265 (95% CI [1.584-3.239]); M:P = 0.006, HR = 2.143 (95% CI [1.251-3.672])), and higher pathological stage (P < 0.001, HR = 2.609 (95%CI [1.911-3.561])) were also associated with poor OS. We then performed a multivariate analysis with the Cox regression model. Due to the missing data of ALK mutation status and M stage over 20%, they were not included in the multivariate analysis. The result indicated that CANT1 expression (P = 0.015, HR = 1.490 (95%CI [1.081-2.055])), T stage (P = 0.032, HR = 1.467(95% CI [1.034-2.081])) and pathological stage (P = 0.001, HR = 2.290 (95% CI [1.395-3.758])) were independently associated with worse OS.

A nomogram integrating the expression of CANT1, T stage, and pathological stage was constructed in our study (Fig. 3A). A worse prognosis was represented by a higher total number of points on the nomogram. The C-index was 0.64 based on 1000 bootstrap replicates for the nomogram. The deviation correction line in the calibration plot was close to the ideal curve, showing that the prediction result is in good agreement with the observation results (Fig. 3B-D).

The GO enrichment items were engaged in several functional groups (Fig. 4A-C): cellular component (8 items), biological process (16 items), and molecular function (8 items). The cellular components for CANT1 and its most similar genes were mainly involved in cell-cell junction, cell cortex, and cell body. The biological processes were enriched in cytoskeleton-dependent cytokinesis, neutrophil-mediated immunity, Ras protein signal transduction, apoptotic signaling pathway, and mitotic nuclear division. The molecular functions were mainly engaged in GDP binding and Ras GTPase binding. Figure 5A showed that an interactive network of the top 19 enrichment terms. It is colored by cluster-ID. Distinct colors are various enrichment pathways of CANT1 correlated genes.

The TCGA RNA-seq data was used to compare CANT1-high and CANT1-low groups’ gene expression in LA. Finally, there are 19,479 differentially expressed genes were identified between the CANT1-high group and CANT1-low group. In order to gain further insight into the biologic pathways involved in LA by the expression level of CANT1, all the differentially expressed genes were analyzed by GSEA. The significantly enriched signaling pathways, mainly including depurination, DNA methylation, and DNA damage telomere stress-induced senescence are enriched based on normalized enrichment score (NES), adjusted P-value, and FDR value (Fig. 5B).

Figure 6A showed that the methylation level was significantly negatively correlated with CANT1 expression with Spearman up to − 0.2561 (P < 0.001). The effect of hypomethylation level and the expression of CANT1 on prognosis in LA was obtained from MethSurv. We discovered that 3 CpG sites located on the CpG island indicated a poor prognosis, including cg00902147, cg16337457, and cg18186771 (Fig. 6B-D).

We constructed A549 and H1299 cells by endogenously knocking down CANT1 by transfection of specific siRNAs, named as A549 si-CANT1 and H1299 si-CANT1. The expression of CANT1 was silenced by three siRNAs, as result, the qRT-PCR assay showed that si-1173 and si-273 had the best silencing effect among the three siRNAs against CANT1 in A549 and H1299 cell lines, respectively (Fig. 7A-B). The results indicated that si-1173 and si-273 were the most effective to inhibit the expression of CANT1 in A549 and H1299 cell lines, respectively. Therefore, they were selected in subsequent experiments.

To explore the effects of CANT1 on LACs’ proliferation and invasion, a series of in vitro experiments were carried out. The results of CCK-8 assays showed significant decline in the proliferation of A549 and H1299 cells after the knockdown of CANT1 (P < 0.001) (Fig. 7C-D). Transwell assay was conducted to evaluate the effects of CANT1 on cell invasion. CANT1 knockdown in A549 and H1299 cells remarkably inhibited the invasion ability (P < 0.001) (Fig. 8).

To better understand the mechanisms of cell proliferation inhibited, the percentages of cells in different phases of the cell cycle were analyzed by flow cytometry. A significant increase in the G1 phase was observed in the si-CANT1 group (69.35 and 82.16%), compared with the NC group and si-NC group (62.41 and 69.65%, 62.43% and 71.66, respectively) in A549 and H1299 cells. Cell cycle was arrest in the G1 phase in the Si-CANT1 group, resulting in less cells entering the S phase (Fig. 9). Summarized, these results confirmed that knockdown of CANT1 could significantly inhibit the malignant phenotypes of LACs.