NPTX1 promotes metastasis via integrin/FAK signaling in gastric cancer.

PURPOSE: This study aimed to investigate the effect of NPTX1 on the prognosis of gastric cancer (GC), as well as the metastatic process in GC.
MATERIALS AND METHODS: The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases were used to analyze the association between NPTX1 expression and prognosis in GC. Quantitative real-time polymerase chain reaction and Western blots were applied to examine the expression of NPTX1 in GC cell lines and expression of genes in downstream pathways. The role of NPTX1 on the migration, invasion, adhesion, and proliferation of GC cell lines was investigated with the transwell assay, the adhesion assay, and the MTT assay. Immunofluorescence staining was used to observe the effect of NPTX1 knockdown on the morphology of cells.
RESULTS: According to the review of TCGA and GEO databases of GC, we found that the expression of NPTX1 increased in cancer tissues and high NPTX1 expression was correlated with poor overall survival, which was associated with lymph node stage in clinicopathologic parameters. Knockdown of NPTX1 attenuated the migration, invasion, and adhesion abilities of GC cells. According to gene set enrichment analysis, NPTX1 was found to be positively related to integrin and focal adhesion (FA). Additionally, NPTX1 knockdown decreased the expression of integrin α1 and integrin α7, followed by deregulation of the expression of p-Src, p-Akt, p-Erk, MMP2, and MMP7, as well as inhibiting the formation of FA complexes and decreasing the length of pseudopods in GC cells.
CONCLUSION: Our study provides strong evidence that NPTX1 plays a crucial role in promoting metastasis and acts as a prognostic indicator in GC.

Introduction

Gastric cancer (GC) is one of the most common cancers, and it has a high mortality rate worldwide.1 Despite the declining incidence of GC during the last several decades, it still remains the third leading cause of cancer-related death.2 Although the development of treatments for GC has been rapid, effective therapeutics for advanced GC have undergone little progress, and median overall survival (OS) is still shorter than 1 year.3 Hence, it is important to diagnose GC as early as possible for patients with advanced disease. However, although many biomarkers have been developed for the prognostic prediction of advanced GC patients, few can be used in clinical practice. Therefore, identifying more sensitive and specific GC-related biomarkers for early diagnosis of GC remains an urgent need.

NPTX1, a member of the pentraxins family, was first identified in the central nervous system and was reported as a risk factor in hypoxic–ischemic neuronal injury4 and amyloid β-induced neuronal loss.5 In recent years, NPTX1 was also found to have low expression in multiple cancers, including lung,6 colon,7 pancreatic,8 and cervical9 cancers, due to the aberrant methylation of 5′ CpG islands in the NPTX1 promoter region. In colon cancer, the decrease in NPTX1 could inhibit cell proliferation by downregulating cyclin A2 and CDK2.7 However, except for the few cancers mentioned above, the expression and functions of NPTX1 in other cancers, including GC, have not yet been defined. Recently, a study about human pluripotent stem cells attracted our attention: high expression of NPTX1 was closely related to the regulation of cell migration, which is also one of the main features of cancers.10 Therefore, we speculated that NPTX1 might play a critical role in promoting cancer metastasis.

In this study, which is based on the reviews of The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases of GC, we interestingly found that the expression of NPTX1 was higher in cancer tissues than in normal tissues and patients with high expression of NPTX1 had significantly shorter OS. Further investigation demonstrated that NPTX1 promoted metastasis via integrin/FAK signaling in GC cells. Our findings suggest that NPTX1 could promote GC progression and might be an important prognostic marker for metastatic and advanced GC.

Materials and methods

Data sources and processing

A total of 351 GC patients, including mRNA sequencing and clinical data, were obtained from TCGA (https://cancerge-nome.nih.gov/). Gene expression profiles of GSE13861 and GSE62254 from GEO database were also examined, which included 90 and 300 GC samples, respectively.11,12 The expression data of NPTX1 and integrin family were collected for each case and NPTX1 was divided into high- and low-expression groups by median.

Cell lines

The GC cell lines BGC823 (TCHu11), MGC803 (TCHu84), and SGC7901 (TCHu46), HGC27 (TCHu22) were obtained from the Chinese Academy of Sciences (Shanghai, China). SNU216 (KCLB00216) was from the Korea Cell Line Bank (KCLB, Seoul, Korea). MKN7 (JCRB1025) and MKN74 (JCRB0255) were from the Japanese Collection of Research Bioresources (JCRB Cell Bank, Osaka, Japan). All the cells were cultured in RPMI-1640 medium containing 10% heat-inactivated FBS at 37°C under an atmosphere of 95% air and 5% CO2.

Antibodies and reagents

Rabbit anti-NPTX1 (20656-1-AP) was purchased from Proteintech (Chicago, IL, USA). Rabbit anti-Akt (#9272S), anti-phospho-Akt (Ser473) (#9271L), anti-phospho-Erk (Thr202/Tyr204) (#4370S), anti-Src (#2110S), anti-phospho-Src (#6943S), anti-FAK (#3285S), anti-phospho-FAK (Y925) (#3284), anti-phospho-FAK (Y397) (#3281S), anti-MMP9 (#3852S), anti-GAPDH (#2118), and anti-integrin β1 (#9699S) were purchased from Cell Signaling Technology (Danvers, MA, USA). Rabbit anti-integrin α7 (ab182941) was purchased from Abcam (Cambridge, MA, USA). Mouse anti-integrin α1 (MAB5676) was from R&D systems (Minneapolis, MN, USA). Mouse anti-ERK (292838), anti-MMP2 (13595), secondary goat anti-rabbit and goat anti-mouse antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Rhodamine phalloidin was obtained from Invitrogen (R415, Waltham, MA, USA). Collagen and laminin were obtained from Sigma (Sigma-Aldrich Co., St Louis, MO, USA). Matrigel was purchased from Corning (Corning Life Science, Tewksbury, MA, USA).

RNA isolation and quantitative real-time PCR

Total RNA from the cultured cells was isolated with Trizol reagent (Invitrogen, Carlsbad, CA, USA) and the concentration of the total RNA was quantified by measuring the absorbance at 260 nm. All reagents for the reverse transcription (RT) were obtained from TaKaRa (Shiga, Japan). The PrimeScript™ RT Reagent Kit (Takara, Japan) was used for mRNA RT. Quantitative real-time PCR was performed using SYBR Premix Ex Taq II (TaKaRa) and measured in Applied Biosystems® 7500 Real-Time PCR Systems (Thermo Fisher Scientific, Waltham, MA, USA). The internal control used was 18S. The 2−ΔΔCt method was used to calculate the fold change of the RNA expression of one sample compared to the calibration sample. The PCR primers used were as follows: NPTX1 forward: 5′-ACCGAGGAGAGGGTCAAGAT-3′; NPTX1 reverse: 5′-GTGGGAATGTGAGCTGGAAC-3′; 18S forward: 5′-CCCGGGGAGGTAGTGACGAAAAAT-3′; 18S reverse: 5′-CGCCCGCCCGCTCCCAAGAT-3′.

RNA interference

NPTX1-specific siRNAs (Si1 and Si2) and negative control siRNA (NC) were prepared by ViewSolid Biotech (Beijing, China). The coding strand of human NPTX1 si1 was 5′-UCCAACAUCUGGGACCGCAATT-3′, and that of NPTX1 si2 was 5′-GCCAACUCGGGCAAACUUUTT-3′. siRNAs were transfected into cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol.

Lentivirus construction and transfection

Lentivirus construction of NPTX1 overexpression carrying green fluorescent protein (GFP) was designed and provided by Obio Technology Corp., Ltd. (Shanghai, China). HGC27 cells were transfected with the lentiviral vector according to the manufacturer’s instructions. Control cells were transfected with empty vector carrying GFP. The NPTX1 overexpression cells were named OE, and empty vector cells, named as NC, were used as control.

MTT assay

BGC823 and SNU216 cells transfected with the NPTX1 siRNA or NC siRNA were incubated in 96-well plates in a density of 5,000 cells/well for 24 hours. Then, 20 µL of the MTT reagent (5 mg/L) was added per well and incubated for another 4 hours. The supernatant was removed and 200 µL of dimethylsulfoxide was added. The absorbance (A) was measured at 570 nm.

Transwell assay

The transwell assay was performed using 8µm-transwell chambers (Corning Life Science). For the invasion assay, the filters were precoated with 50 µL matrigel at 1:4 dilution in serum-free RPMI-1640 to form a genuine reconstituted basement, then 500 µL RPMI-1640 medium containing 10% FBS was added to the lower chamber. For the migration and invasion assays, BGC823 and SNU216 were placed into the upper chamber at a density of 2×104 cells/200 µL and 5×104 cells/200 µL, respectively. After incubation for 24 hours, cells remaining on the upper membrane were removed with a cotton swab, while cells that had invaded through the membrane were fixed in methanol, stained with Wright–Giemsa. Five representative fields of each insert were randomly counted under a microscope (Olympus, Tokyo, Japan) and analyzed statistically.

Adhesion assay

BGC823 and SNU216 cells transfected with NPTX1 siRNA or NC siRNA were plated in 96-well plates which were precoated with 10 µg/mL collagen, laminin, or matrigel overnight at 37°C, at a density of 1×104 cells/well with serum-free RPMI-1640. After incubating at 37°C for 30 minutes and 60 minutes, cells that did not adhere to the plates were washed off with PBS. Adherent cells were fixed in 4% paraformaldehyde, stained with Wright–Giemsa, and counted at five random fields under a microscope (Olympus) and analyzed statistically.

Western blot

Western blot was performed as previously described.13 After solubilizing the cells in 1% Triton X-100 lysis buffer, protein lysates were separated by SDS-PAGE and electrophoretically transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, Bedford, MA, USA). The membranes were blocked with 5% skim milk in tris-buffered saline and Tween 20 (TBST) buffer at room temperature and incubated overnight at 4°C with the indicated primary antibodies. After the appropriate secondary antibodies were added at room temperature, the proteins were detected with enhanced chemiluminescence reagent and visualized with the Electrophoresis Gel Imaging Analysis System (DNR Bio-Imaging Systems, Jerusalem, Israel).

Cell spreading assay and immunofluorescence microscopy

Cell spreading assay was performed as described previously with some modification.14 Cell culture dishes (Cat. No.801001, Nest, China) were coated with matrigel, followed by blocking with 1% BSA/PBS. The siRNA-transfected cells were detached with 0.05% EDTA/PBS, and 4×104 cells were seeded per chamber and incubated at 37°C. After 4 hours, the cells were fixed in 4% paraformaldehyde for 30 minutes, permeabilized with 0.2% Triton X-100 for 15 minutes and blocked with 5% BSA for 2 hours. For staining, the cells were primed with anti-FAK and phalloidin for 1 hour at room temperature and then overnight at 4°C. The next day, rabbit antibody (Alexa Fluor 488) was added for 2 hours at room temperature in the dark and the cells were mounted using the SlowFade Antifade Kit. Finally, cells were observed and imaged by CQ1 spinning disk confocal systems (Yokogawa Electric Corp., Tokyo, Japan).

Gene set enrichment analysis (GSEA)

The software GSEA v2.2.3 (www.broadinstitute.org/gsea) was used to perform GSEA. NPTX1 expression level was dichotomized as low and high categories to annotate phenotype, and Kyoto Encyclopedia of Genes and Genomes (KEGG) gene sets from Molecular Signatures Database (MSigDB) were used as functional gene sets. P<0.05 and false discovery rate (FDR) <0.25 were utilized as threshold values to estimate the statistical significance.

Statistical analysis

Data were confirmed in three independent experiments and the presented data are shown as mean ± SD. Correlation of NPTX1 with clinicopathological parameters was evaluated by chi-squared test. The correlation coefficients were determined using Pearson’s correlation test. Survival curves were determined using the Kaplan–Meier method and compared using the log-rank test. Univariate and multivariate analyses were performed by Cox proportional hazards regression models to estimate HR and 95% CI. Clinicopathological parameters significantly associated with OS in the multivariate Cox regression model were identified and integrated as prognostic variables. A nomogram was established by R software with the discrimination and calibration assessed by concordance index (c-index) and calibration curve, respectively. P<0.05 was considered statistically significant. All statistical analyses were conducted using the SPSS statistical software package (version 16.0), GraphPad Prism 7 and R software (V 3.3.3).

Results

High expression of NPTX1 predicts poor prognosis for GC patients

To determine the expression of NPTX1 in GC, the mRNA levels of NPTX1 were obtained from the GSE13861 dataset, which contained 19 normal human gastric tissues and 71 GC tissues. The expression of NPTX1 in GC tissues was higher than in normal tissues (P=0.044, Figure 1A). To analyze whether the expression of NPTX1 affects the prognosis of GC, 351 GC patients with clinical characteristics from TCGA were divided into NPTX1 high- and low-expression groups. As shown in Table 1, high expression of NPTX1 was associated with older age (P=0.031) and poor lymph node stage (P=0.015). Kaplan–Meier analysis showed that OS in patients with high NPTX1 expression was significantly shorter than in patients with low NPTX1 expression (14.7 vs 18.5 months, P=0.015; Figure 1B), indicating that NPTX1 might be useful for the prediction of clinical prognosis in GC. The univariate analysis showed that shorter OS was correlated with age older than 60 years, lymph node metastasis, distant metastasis, and high NPTX1 expression. Further, the multivariate Cox regression analysis showed that NPTX1 expression (HR =1.451, 95% CI =1.034–2.036, P=0.031), age older than 60 years (HR =1.796, 95% CI =1.226–2.632, P=0.003), lymph node metastasis (HR =1.755, 95% CI =1.149–2.680, P=0.009), and distant metastasis (HR =2.473, 95% CI =1.422–4.302, P=0.001) were independent predictors for poor OS (Table 2). On the basis of the multivariate Cox regression model, a nomogram was constructed to predict 1-, 2-, and 3-year OS (Figure 1C). The c-index for examining the predictive accuracy of 1-, 2-, and 3-year OS was 0.658 in this cohort (95% CI =0.634–0.683). Moreover, the red lines, which represent the predicted values, fluctuated above and below the gray lines, which represent the actual values, indicating that the predictive capability of this nomogram for OS was reliable (Figure 1D). Similar results were also obtained from the GSE62254 validation cohort (Figure 1E and Tables S1 and S2). Taken together, these results suggest that high expression of NPTX1 is associated with poor prognosis and that NPTX1 might be involved in the metastatic process and the promotion of GC progression.

Figure 1

NPTX1 was elevated in GC tissues and associated with poor prognosis in GC.

Notes: (A) NPTX1 expression analysis in GC tissues (n=71) and normal tissues (n=19) by using GSE13861. (B) Kaplan–Meier analysis for the OS of GC patients in TCGA dataset. (C) Nomogram predicting 1-, 3-, and 5-year OS for GC patients from TCGA dataset. (D) Calibration curve to validate nomogram for 1- and 3-year OS. (E) Kaplan– Meier curve for the OS of GC patients in GSE62254 dataset.

Abbreviations: GC, gastric cancer; OS, overall survival; TCGA, The Cancer Genome Atlas.

Table 1

Association of NPTX1 expression with the GC clinicopathological characteristics in TCGA cohort

	N (351)	NPTX1 expression level		P-value
		Low [n (%)]	High [n (%)]
Gender				0.373
Female	125	58 (46.4)	67 (53.6)
Male	226	117 (51.8)	109 (48.2)
Age (years)				0.031
≤60	116	48 (41.4)	68 (58.6)
>60	235	127 (54.0)	108 (46.0)
Tumor stage				0.07
T1–T3	257	136 (52.9)	121 (47.1)
T4	94	39 (41.5)	55 (58.5)
Lymph node stage				0.015
N0	107	64 (59.8)	43 (40.2)
N1–N3	244	111 (47.6)	133 (57.1)
Metastasis stage				0.542
M0	326	161 (49.4)	165 (50.6)
M1	25	14 (56)	11 (44)

Note: Bold values indicate statistical significance, P<0.05.

Abbreviations: GC, gastric cancer; TCGA, The Cancer Genome Atlas.

Table 2

Univariate and multivariate Cox regression of prognostic factors for OS in GC in TCGA cohort

	Univariate analysis			Multivariate analysis
	P-value	HR	95% CI	P-value	HR	95% CI
Age (years)	0.03	1.509	1.041–2.188	0.003	1.796	1.226–2.632
Gender	0.193	1.266	0.887–1.805
Tumor stage	0.361	1.185	0.823–1.705
Lymph node stage	0.01	1.959	1.297–2.961	0.009	1.755	1.149–2.680
Metastasis stage	0.002	2.341	1.369–4.004	0.001	2.473	1.422–4.302
NPTX1	0.016	1.501	1.077–2.092	0.031	1.451	1.034–2.036

Note: Bold values indicate statistical significance, P<0.05.

Abbreviations: GC, gastric cancer; OS, overall survival; TCGA, The Cancer Genome Atlas.

NPTX1 promotes migration, invasion, and ECM adhesion in GC cells

To demonstrate whether NPTX1 could promote GC metastasis, the effect of NPTX1 on migration, invasion, and cell– extracellular matrix (ECM) adhesion was performed using GC cells. After comparing the expression of NPTX1 among different GC cells (MKN7, SNU216, MKN74, MGC803, BGC823, SGC7901, and HGC27) by Western blot (Figure 2A), BGC823 and SNU216 cells were selected for the loss of function experiments and HGC27 cells were selected for the gain of function experiments. In BGC823 and SNU216 cells with NPTX1 siRNAs (Si1 and Si2) and NC siRNA, the results of transwell assays showed that the migration and invasion abilities of GC cells were significantly decreased in NPTX1-knockdown cells (P<0.01, Figure 2B, C); no significant difference in proliferation ability was observed between NC and NPTX1 siRNA cells (Figure S1). In addition, knockdown of NPTX1 significantly decreased cells’ abilities to adhere to ECM (P<0.01, Figure 2D). In contrast, overexpression of NPTX1 in HGC27 cells with NPTX1 lentivirus infection increased the cells’ abilities for migration and invasion (Figure S3C). The data suggest that NPTX1 facilitates the process of metastasis in GC cells.

Figure 2

NPTX1 promotes migration, invasion, and adhesion in GC cells.

Notes: (A) Western blot of NPTX1 expression in GC cell lines. (B) Western blot was used to detect NPTX1 expression in BGC823 and SNU216 cells transfected with the siNC or the siNPTX1. (C) Transwell migration and invasion assays of BGC823 and SNU216 with transient NPTX1 knockdown (up panels). Quantifications of cells are shown as proportions of the number of control cells (down panels). Original magnification, 100×. Scale bar: 200 µM. (D) Adhesion assay of BGC823 and SNU216 with transient NPTX1 knockdown (up panels). Quantifications of cells are shown as proportions of the number of control cells (down panels). Original magnification, 200×. Scale bar: 100 µM. *P<0.05. GAPDH was used as a loading control in Western blot. Error bars represent the mean ± SD of three independent experiments.

Abbreviations: GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GC, gastric cancer; NC, negative control.

NPTX1 promotes metastasis by interacting with integrins in GC cells

To further clarify the underlying mechanisms of NPTX1 in the metastatic process, GSEA using the TCGA GC dataset was performed. As shown in Figure 3A, NPTX1 expression was positively correlated with multiple cancerous functions, especially the functions of ECM–receptor interaction and focal adhesion (FA). Subsequently, 33 genes, which were identified in the functions of both ECM–receptor interaction and FA, were screened using the VENNY tool (Figure 3B). Among these genes, the integrin family (ITG), which is known to play an extremely important role in both FA and cancer-related metastasis,15 was chosen for the further mechanism study. With correlation analysis, the expression of NPTX1 was shown to be positively related to the expression of ITG genes: ITGA1 (r=0.5477, P<0.0001), ITGA7 (r=0.5518, P<0.0001), ITGA8 (r=0.1919, P<0.0001), ITGA9 (r=0.4498, P<0.0001), ITGA11 (r=0.1691, P=0.0006), ITGAV (r=0.1544, P=0.0018), ITGB1 (r=0.23, P<0.0001), ITGB3 (r=0.1946, P<0.0001), and ITGB5 (r=0.07584, P=0.1266; Figure 3C). Similar results were also observed in the GSE62254 dataset (Figure S2). These data indicate that NPTX1 might promote metastasis by enhancing focal contacts through the ITG family. The top two genes according to the correlation index, ITGA1 (integrin α1) and ITGA7 (integrin α7), were chosen for further study.

Figure 3

NPTX1 is involved in focal contacts.

Notes: (A) The enrichment of genes involved in ECM receptor and FA in NPTX1-high GC. (B) Venn diagram analysis of the coexpression genes in set ECM receptor and set FA. (C) Correlation of NPTX1 and integrin subunits in human GC tissues based on TCGA dataset.

Abbreviations: ECM, extracellular matrix; FA, focal adhesion; GC, gastric cancer; TCGA, The Cancer Genome Atlas; NES, normalized enrichment score; FDR, false discovery rate; KEGG, Kyoto Encyclopedia of Genes and Genomes.

NPTX1 enhances cell–matrix adhesion by affecting focal contacts formation through integrin/FAK signaling

It is well known that the binding of integrin α1 and integrin α7 to their specific ECM proteins, collagen and laminin, respectively, initiates the formation of FA complexes, which are composed of series of proteins such as paxillin, Src, FAK, and talin, and play an important role in cancer cell migration.16,17 Therefore, we examined whether NPTX1 could promote FA formation through integrin α1 and integrin α7 in GC cells. As a result, knockdown of NPTX1 not only significantly decreased the ability of cells to adhere to collagen and laminin (Figure 4A), but also dramatically downregulated the expressions of ITGA1 and ITGA7 and the levels of p-FAK (Tyr397), p-FAK (Tyr925), p-Src, p-Akt, and p-Erk, as well as the downstream genes, MMP2 and MMP9 (Figure 4B). Conversely, overexpression of NPTX1 in HGC27 cells increased the cells’ abilities to adhere to collagen and laminin and upregulated the expression of ITGA1, ITGA7, p-FAK (Tyr397), p-FAK (Tyr925), p-Src, p-Akt, p-Erk, MMP2, and MMP9 (Figure S3D, E). Further, the effect of NPTX1 on the morphology and length of FAs, which were stained with the FAs marker FAK,14 was observed by confocal microscopy. As shown in Figure 4C, compared with NC cells in which FAK was localized on the leading edge at the periphery of cells, there were fewer pseudopods and the length of FAs was shorter in NPTX1-knockdown cells, indicating that NPTX1 promotes the formation of FAs in the leading edge of GC cells. These results strongly suggest that NPTX1 promotes metastasis in GC cells by facilitating the formation of focal contacts through integrin/FAK signaling.

Figure 4

NPTX1 facilitates focal contacts via integrin/FAK pathway.

Notes: (A) Adhesion assay of BGC823 and SNU216 with transient NPTX1 knockdown (up panels). Quantifications of cells are shown as proportions of the number of control cells (down panels). Original magnification, 200×. Scale bar: 100 µM. *P<0.05. Error bars represent the mean ± SD of three independent experiments. (B) Focal contacts-related genes were assessed by Western blot in BGC823 and SNU216 cells transfected with siNPTX1; GAPDH was used as a loading control. (C) The effects of NPTX1 knockdown on the morphology and length of FA complexes were determined by immunofluorescence assay. Original magnification, 400×. *P<0.05.

Abbreviations: FAK, focal adhesion kinase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; FA, focal adhesion.

Discussion

In the present study, we found that NPTX1 expression was elevated in GC tissues compared with normal tissues, and NPTX1 was positively associated with poor prognosis in GC patients. Importantly, NPTX1 was confirmed to play a critical role in the metastatic process of GC cells by promoting focal contacts via the integrin/FAK pathway.

According to the analysis of the GSE13861 dataset, we found that the expression of NPTX1 was higher in GC tissues than in normal tissues, which was inconsistent with previous reports.6,8 Previous studies reported that the expressions of NPTX1 in the tissues of lung cancer and pancreatic cancer were lower than in normal tissues because of methylation, but the methylation status of NPTX1 was not significantly different between cervical cancer and normal tissues.18 These contradictory results might account for the different methylation statuses in different cancer types. In patients with lung cancer who have undergone radical surgery, the OS in patients with methylated NPTX1 was shorter than in those with unmethylated NPTX1.6 Here, our results, which are based on TCGA and GSE62254 datasets, showed that OS in GC patients with high NPTX1 expression was significantly shorter than in those with low NPTX1 expression. Further study is needed to investigate the methylation status of NPTX1 and the protein expression in the tissues of GC and other kinds of cancers in order to prove the important role of NPTX1 in cancer development.

The finding that NPTX1 expression was positively related to lymph node stage, suggested that NPTX1 might promote GC metastasis. We proved with the in vitro experiments that overexpression of NPTX1 promoted the abilities of migration, invasion, and adhesion of GC cells. Our GSEA results showed that high expression of NPTX1 was correlated with ECM–receptor interaction and FA, which is similar to a recent report that high expression of NPTX1 was closely related to the regulation of cell migration in human pluripotent stem cells using gene ontology analysis.10 These analyses provided a directional clue for the mechanisms of NPTX1 in promoting GC metastasis. Therefore, ITG family members ITGA1 and ITGA7, which were involved in both ECM–receptor interaction and FA, as well as highly correlated with NPTX1, were chosen for the investigation of underlying mechanisms of NPTX1 in the metastatic process.

Integrin α1 and integrin α7 are known to facilitate cell– ECM adhesion through interaction with collagen and laminin, respectively.19 In the present study, NPTX1 not only promoted the ability of GC cells to adhere to collagen and laminin but also increased the expressions of ITGA1 and ITGA7. On the other hand, after binding to their own specific ECM protein, the activation of integrin subunits could lead to the formation and maturation of FAs, in which FAK is recruited rapidly and phosphorylated at the Y397 site, and subsequent activation of vinculin and paxillin.20–22 The autophosphorylation of FAK at the Y397 site creates a binding site for the phosphorylation of Src at Y416 and, further, Src phosphorylates FAK at Y925, which is necessary for FA turnover by promoting FAs disassembly and formation of protrusions.23–26 Similarly, our results showed that after ITGA1 and ITGA7 were downregulated by NPTX1 knockdown, the levels of p-FAK (Y925/Y397), p-Src, p-Akt, and p-Erk, as well as their downstream genes MMP2 and MMP9, were all decreased. Knockdown of NPTX1 also inhibited the formation of FAs at the top of the cells’ periphery. We also demonstrated that NPTX1 promoted GC metastasis by upregulating the expressions of ITGA1 and ITGA7 and increasing focal contacts formation via the integrin/FAK pathway.

Focal contacts mediate several cell processes, including the epithelial–mesenchymal transition (EMT) process, which is necessary for cancer cells to migrate and invade through the ECM.27 The EMT process is often accompanied by reduced cell–cell adhesions and strengthened cell–ECM adhesions.28 Here, we demonstrated that NPTX1 facilitated the activation of FAK and Src, which are pivotal kinases in focal contacts. Therefore, we speculated that NPTX1 might promote metastasis through the EMT process. The underlying mechanisms of NPTX1 regulating the EMT process need to be studied further.

In summary, this study revealed, for the first time, that the high expression of NPTX1 leads to poor clinical outcomes through integrin α1/7-FAK signaling-mediated metastasis in GC. NPTX1 might have potential value as a diagnostic and prognostic indicator for GC.

The viability of cells during migration and invasion was carried out by MTT assays.

Abbreviations: NC, negative control; n.s, no significance.

Correlation of NPTX1 and integrin subunits in human GC tissues based on GSE62254 dataset.

Overexpression of NPTX1 promotes migration, invasion, and adhesion through integrin/FAK pathway in GC cell.

Notes: (A) Western blot and PCR were used to detect NPTX1 expression in HGC27. (B) Transwell migration and invasion assays of HGC27 with NPTX1 overexpression (left panels). Quantifications of cells are shown as proportions of the number of control cells (right panels). Original magnification, 100×. Scale bar: 200 µM. (C) The viability of HGC27 cells during migration and invasion was carried out by MTT assays. (D) Adhesion assays of HGC27 cells with NPTX1 overexpression (up panels). Quantifications of cells are shown as proportions of the number of control cells (down panels). Original magnification, 200×. Scale bar: 100 µM. (E) Focal contacts-related genes were assessed by Western blot in HGC27 cells with NPTX1 overexpression. *P<0.05. GAPDH was used as a loading control in Western blot. Error bars represent the mean ± SD of three independent experiments.

Abbreviations: FAK, focal adhesion kinase; GC, gastric cancer; NC, negative control; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; OE, overexpression; n.s, no significance.

Table S1

Association of NPTX1 expression with the GC clinicopathological characteristics in GSE62254 cohort

	N (300)	NPTX1 expression level		P-value
		Low [n (%)]	High [n (%)]
Gender				0.014
Female	101	40 (39.6)	61 (60.4)
Male	199	110 (55.3)	89 (44.7)
Age (years)				0.018
≤60	117	48 (41.0)	69 (59.0)
>60	183	102 (55.7)	81 (44.3)
Tumor stage				0.022
T1–T3	279	145 (52.0)	134 (48.0)
T4	21	5 (23.8)	16 (76.2)
Lymph node stage				0.011
N0	38	25 (65.8)	13 (34.2)
N1–N3	262	125 (47.7)	137 (52.3)
Metastasis stage				0.158
M0	273	140 (51.3)	133 (48.7)
M1	27	10 (37.0)	17 (63.0)

Note: Bold values indicate statistical significance, P<0.05.

Abbreviation: GC, gastric cancer.

Table S2

Univariate and multivariate Cox regression of prognostic factors for OS in GC in GSE62254 cohort

	Univariate analysis			Multivariate analysis
	P-value	HR	95% CI	P-value	HR	95% CI
Age (years)	0.168	1.263	0.906–1.761
Gender	0.559	1.105	0.790–1.545
Tumor stage	0.02	1.85	1.1–3.109
Lymph node stage	<0.01	3.427	1.604–7.322	0.009	2.772	1.287–5.968
Metastasis stage	<0.01	3.837	2.480–5.938	<0.01	3.101	1.968–4.885
NPTX1	<0.01	1.674	1.209–2.316	0.047	1.402	1.005–1.957

Note: Bold values indicate statistical significance, P<0.05.

Abbreviations: GC, gastric cancer; OS, overall survival.