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The cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING)-mediated senescence-associated secretory phenotype (SASP) pathway has recently been identified in the suppression and promotion of cancers. However, its practical role in carcinogenesis remains to be comprehensively elucidated. Here, we describe an investigation analysing SASP activity and its correlations with DNA damage response (DDR), genomic mutations, and cell proliferation in gastric carcinogenesis among 30 cases with available endoscopic submucosal dissection (ESD) specimens of early neoplastic lesions (including low-grade dysplasia [LGD], high-grade dysplasia [HGD], and intramucosal carcinoma). The positive cells of senescence-associated β-galactosidase staining and cGAS, STING, interferon-regulatory factor 3 (IRF3), and signal transducer and activator of transcription 6 (STAT6) expression levels using immunostaining were elevated in HGD and in cancers. Similarly, increased expression of the Fanconi anemia group D2 (FANCD2) protein, tumour suppressor p53 binding protein 1 (TP53BP1), and replication protein A (RPA2) (i.e., primary DDR factors) was detected in HGD and in cancers; these increased expression levels were closely correlated with high expression of Ki67 and minichromosome maintenance complex component 7 (MCM7) proteins. Moreover, genomic mutations in
Senescent cells can produce pro-inflammatory factors, including cytokines, growth factors, proteases, and chemokines; this state is collectively termed the senescence-associated secretory phenotype (SASP) [
Gastric cancers are widely distributed throughout the world, and are especially prevalent in East Asian countries [
In the present investigation, ESD specimens from patients with early gastric cancer and precancerous lesions were evaluated. SASP factors, DNA damage response (DDR), genetic mutations, and cell proliferation were analysed to determine the role of SASP as well as its correlations with DDR,
Thirty ESD specimens from patients with early gastric cancer were selected from all ESD specimens available at the Department of Pathology (Peking University First Hospital) from 2016 to 2018. Clinical information for these specimens was abstracted from patient medical records. In addition, five fresh ESD specimens were collected. Pathological evaluation was conducted based on the 5th World Health Organization classification system [
The distribution of lesions in 30 cases of ESD specimens.
Case NO. | LGD | HGD | Cancer | Differentiation | Stage |
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1 | √ | √ | Well | pT1b1 | |
2 | √ | √ | √ | Well | pT1a |
3 | √ | √ | √ | Well | pT1b1 |
4 | √ | √ | Poor | pT1b2 | |
5 | √ | √ | √ | Poor | pT1a |
6 | √ | Poor | pT1b1 | ||
7 | √ | √ | √ | Well | pT1a |
8 | √ | √ | √ | Well | pT1b1 |
9 | √ | √ | Well | pT1a | |
10 | √ | √ | √ | Well | pT1b2 |
11 | √ | Poor | pT1b1 | ||
12 | √ | √ | √ | Well | pT1a |
13 | √ | √ | √ | Well | pT1a |
14 | √ | √ | √ | Well | pT1a |
15 | √ | √ | Well | pT1a | |
16 | √ | √ | √ | Well | pT1a |
17 | √ | √ | √ | Well | pT1b2 |
18 | √ | √ | √ | Poor | pT1a |
19 | √ | √ | √ | Well | pT1a |
20 | √ | √ | √ | Poor | pT1b1 |
21 | √ | √ | √ | Poor | pT1b1 |
22 | √ | √ | √ | Well | pT1b2 |
23 | √ | √ | √ | poor | pT1a |
24 | √ | √ | √ | Well | pT1a |
25 | √ | √ | √ | Well | pT1a |
26 | √ | √ | Well | pT1a | |
27 | √ | √ | Well | pT1a | |
28 | √ | √ | Poor | pT1a | |
29 | √ | √ | Poor | pT1a | |
30 | √ | √ | Poor | pT1a |
ESD, endoscopic submucosal dissection; HGD, high-grade dysplasia; LGD, low-grade dysplasia.
The 30 included patients comprised 24 male and 6 female patients, with ages ranging from 43 to 79 years (median, 65 years). A total of 12 cases involved the pyloric antrum, whereas the other 18 cases involved the body or fundus. Clinical information and follow-up data were obtained for all cases. The follow-up period was defined as starting from the date of initial diagnosis and ending at the date of the patient’s death, progression, relapse, or last follow-up visit. The follow-up duration ranged from 10 to 54 months (median, 36.5 months). Frozen sections from five additional ESD specimens derived from patients with early gastric carcinomas were subjected to SA-β-gal staining.
Frozen sections from fresh ESD specimens were fixed in 4% formalin and evaluated using an SA-β-gal staining kit (GenMed Scientifics, Inc., Wilmington, DE, United States) according to the manufacturer’s instructions. Briefly, tissues were stained with the SA-β-gal staining solution overnight at 37°C; we counted 100 cells in random fields, and calculated the percentage of SA-β-gal-positive cells (i.e., blue cells).
Formalin-fixed, paraffin-embedded (FFPE) sections were deparaffinized with serial xylene treatments and hydrated in graded alcohols. Endogenous peroxidase activity was quenched using 0.3% hydrogen peroxide for 60 min. Antigen retrieval was carried out by heating the specimen in citrate buffer (20-mM citrate buffer, pH 6.0) at 95°C for 20 min. After blocking with horse serum (1:100 in phosphate buffered saline, PBS), the sections were incubated with primary antibodies in various dilutions overnight at 4°C. The specimens were incubated using a Dako Envision Flex amplification kit (Dako, Glostrup, Denmark) for 60 min, and colour development was completed using a freshly prepared diaminobenzidine solution (Dako). The sections were then counterstained using Mayer’s haematoxylin. For the negative control, the primary antibody was replaced with phosphate buffered saline (PBS) or normal rabbit serum. The relevant antibodies and their associated information are summarized in
We found that cGAS, STING, and IRF3 demonstrated cytoplasmic staining, while STAT6, Ki67, minichromosome maintenance complex component 7 (MCM7), and p53 showed nuclear staining. The evaluation was performed in whole slides. Since the intensive staining for each of these factors was similar, the positive stained glandular epithelial cells were semiquantitatively estimated and the resultant percentages represent the positive glandular epithelial cells against total glandular cells for each of groups. Stromal cells were not counted. For associations with clinicopathological parameters and Kaplan–Meier single-factor analysis, positive cases were defined as those with either >10% or >20% positive tumour cells (i.e., different thresholds were evaluated), while negative cases were defined as those with ≤10% or ≤20% positive tumour cells.
DNA was extracted from FFPE blocks derived from 30 cases using a QIAamp DNA FFPE tissue kit (Qiagen, Hilden, Germany). The DNA library was constructed using the capture method, and paired-end sequencing was performed using a NextSeq 500 Sequencer in combination with the NextSeq™ 500 High Output Kit (Illumina, Inc., San Diego, CA, United States) according to the manufacturer-recommended protocols for gastrointestinal tumour-related genes (Burning Rock, Guangzhou, China). The average sequencing depth was 1,000× for tissue samples. Single nucleotide variants, copy number variants, and fusion were called in the pipeline. Target regions were captured using designed probes spanning 41 genes (
Sequence data were mapped onto the reference human genome (hg19) using Burrows–Wheeler Aligner version 0.7.10. Local alignment optimization, duplication marking, and variant calling were performed using the Genome Analysis Tool Kit version 3.2 and VarScan version 2.4.3.
All statistical analyses were performed using SPSS statistical software (version 17.0, Chicago, IL, United States), Microsoft Excel 2007 (Seattle, WA, United States), and GraphPad Prism software (San Diego, CA, United States). The data obtained from immunohistochemistry (IHC) and association between expressed factors and other parameters were analysed using chi-square tests, Fisher exact tests, Mann–Whitney U tests, and Spearman correlation analysis. The distributions of IHC results between different lesion types were analysed using nonparametric Friedman tests. Progression-free survival curves were plotted using the Kaplan-Meier method and were compared using log-rank tests. Differences were considered statistically significant given a two-sided
Fresh frozen sections from five ESD specimens of early gastric carcinomas were subjected to SA-β-gal staining. Results showed positive staining in the cytoplasm of precancerous and cancerous cells in all evaluated cases (>10% SA-β-gal-positive cells) but not in the inflammatory gastric mucosa (
SASP activity in gastric lesions.
Expression of cGAS, STING, IRF3, and STAT6 factors involved in the mediation of SASP activity was analysed in the 30 ESD specimens. cGAS staining was mainly detected in the cytoplasm of precancerous and cancerous cells (
On performing Spearman correlation, we found that cGAS expression positively correlated with IRF3 and STAT6 expression, but not with STING expression (
Correlations between SASP, DDR, and proliferation factors in early gastric cancer.
STING | IRF3 | STAT6 | FANCD2 | TP53BP1 | RPA2 | Ki67 | MCM7 | |
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cGAS |
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STING |
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IRF3 |
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STAT6 |
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FANCD2 |
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TP53BP1 |
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Ki67 |
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cGAS, cyclic GMP-AMP synthase; FANCD2, Fanconi anemia group D2; IRF3, interferon-regulatory factor 3; MCM7, minichromosome maintenance complex component 7; RPA2, replication protein A; STAT6, signal transducer and activator of transcription 6; STING, cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes; TP53BP1, tumour suppressor p53 binding protein 1.
*Indicates statistical significance. Statistical analyses were carried out using Spearman correlation.
SASP activation is induced by the binding of cGAS to free cytoplasmic DNA generated following DNA damage [
Expression of DDR in gastric lesions.
Mutual correlations were also statistically evaluated using Spearman correlation tests, the results of which indicated statistically significantly positive correlations between FANCD2, TP53BP1, and RPA2 expression, with a closer correlation detected between FANCD2 and TP53BP1 expression (
All cases were subjected to targeted sequencing using a panel of 41 genes expressed in gastric cancers. The detected genomic mutations are summarized in
Case | IHC (%*) | Mutation | Mutation location |
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1 | ND | Splicing | Exon 5, c.376-2A>G |
2 | ND | Nonsense | Exon 4, c.159G>A |
3 | Positive (95%) | Missense | Exon 6, c.641A>G |
4 | Positive (90%) | Missense | Exon 8, c.818G>A |
5 | Positive (95%) | Missense | Exon 7, c.733G>A |
6 | Positive (95%) | Frameshift | Exon 9, c.956del |
7 | Positive (95%) | Missense | Exon 8, c.844C>T |
8 | ND | Nonsense | Exon 8, c.915C>T |
9 | ND | WT | WT |
10 | Positive (95%) | Missense | Exon 7, c.733G>A |
11 | ND | WT | WT |
12 | ND | Frameshift | Exon 5, c.491_428del |
13 | Negative | WT | WT |
14 | ND | WT | WT |
15 | ND | WT | WT |
16 | Negative | Splicing | Intron 9, c.994-1G>A |
17 | Positive (95%) | Missense | Exon 8, c.817C>T |
18 | ND | WT | WT |
19 | Positive (90%) | Missense | Exon 8, c.844C>T |
20 | Negative | WT | WT |
21 | Negative | Frameshift | Exon 5, c.463_466del |
22 | Positive (90%) | Missense | Exon 8, c.818G>A |
23 | Positive (90%) | Missense | Exon 8, c.817C>T |
24 | Negative | WT | WT |
25 | Negative | Frameshift | Exon 7, c.675-5_675dup |
26 | Negative | WT | WT |
27 | Negative | WT | WT |
28 | Negative | WT | WT |
29 | Negative | WT | WT |
30 | ND | WT | WT |
ESD, endoscopic submucosal dissection; HGD, high-grade dysplasia; IHC, immunohistochemistry; ND, IHC not performed; TP53, tumour protein 53; WT, wild-type.
*Percentage of positive cells in HGD and in cancer specimens.
Immunostaining for p53 in gastric lesions and the association between TP53 mutation and SASP, DDR, and proliferation factors in early gastric cancer.
Proliferation zone expansion with an increasing Ki67 index is generally characteristic of gastric carcinogenesis [
Ki67 and MCM7 expression in gastric lesions.
Finally, we found a moderately strong correlation between Ki67 and MCM7 immunostaining (
We performed statistical analyses to clarify correlations between expression levels for SASP, DDR, and proliferation factors in early gastric cancers. The results are summarized in
Next, the expression profile in early gastric cancer was generated based on the expression of SASP, DDR, and proliferation factors in reference to
Heatmap of expression profiling in early gastric cancer, using ESD. Expression profiling was generated based on immunostaining for SASP expression, DDR expression, and cell proliferation in regard to TP53 mutation status. Abbreviations: DDR, DNA damage response; ESD, endoscopic submucosal dissection; SASP, senescence-associated secretory phenotype; TP53, tumour protein 53; WT, wild type. Stars indicate cases with relapse (No. 14, 19, and 24).
To identify the possible biological role of the expression of SASP factors in early gastric cancer, associations between the expression of SASP factors and clinical features were estimated in the present study. In following up on the clinical course of each of the 30 cases, we found that four cases underwent subtotal gastrectomy (three cases) or total gastrectomy (one case) following the ESD procedure. Three patients with early gastric cancer relapsed; these patients were determined to have intramucosal carcinoma (pT1a) and well-differentiated adenocarcinoma (without radical operation).
Associations between the expression of SASP factors and other clinicopathological parameters were analysed using either a >10% or >20% cut-off. Cases with lesions located in the gastric pyloric antrum showed a statistically significantly elevated expression of IRF3, as compared with cases with lesions located in the body/fundus (>10%,
Association between SASP factors and characteristics of patients with early gastric cancer.
Total |
cGAS expression (%) | STING expression (%) | IRF3 expression (%) | STAT6 expression (%) | |||||||||
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Positive | Negative |
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Positive | Negative |
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Positive | Negative |
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Positive | Negative |
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16 (53.3) | 14 (46.7) | 21 (70.0) | 9 (30.0) | 20 (66.7) | 10 (33.3) | 22 (73.3) | 8 (26.7) | ||||||
Gender | |||||||||||||
Male | 24 (80.0) | 13 (43.3) | 11 (36.7) | 1 | 16 (53.3) | 8 (26.7) | 0.637 | 17 (56.7) | 7 (23.3) | 0.372 | 18 (60.0) | 6 (20.0) | 0.645 |
Female | 6 (20.0) | 3 (10.0) | 3 (10.0) | 5 (16.7) | 1 (3.3) | 3 (10.0) | 3 (10.0) | 4 (13.3) | 2 (6.7) | ||||
Age(years) | |||||||||||||
≤60 | 9 (30.0) | 5 (16.7) | 4 (13.3) | 1 | 6 (20.0) | 3 (10.0) | 1 | 6 (20.0) | 3 (10.0) | 1 | 8 (26.7) | 1 (3.3) | 0.374 |
>60 | 21 (70.0) | 11 (36.7) | 10 (33.3) | 15 (50.0) | 6 (20.0) | 14 (46.7) | 7 (23.3) | 14 (46.7) | 7 (23.3) | ||||
Location | |||||||||||||
Body/fundus | 18 (60.0) | 9 (30.0) | 9 (30.0) | 0.722 | 11 (36.7) | 7 (23.3) | 0.249 | 9 (30.0) | 9 (30.0) |
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14 (46.7) | 4 (13.3) | 0.678 |
Antrum/pyloricus | 12 (40.0) | 7 (23.3) | 5 (16.7) | 10 (33.3) | 2 (6.7) | 11 (36.7) | 1 (3.3) | 8 (26.7) | 4 (13.3) | ||||
Differentiation | |||||||||||||
Well | 19 (63.3) | 8 (26.7) | 11 (36.7) | 0.142 | 14 (46.7) | 5 (16.7) | 0.687 | 12 (40.0) | 7 (23.3) | 0.702 | 13 (43.3) | 6 (20.0) | 0.672 |
Poor | 11 (35.7) | 8 (26.7) | 3 (10.0) | 7 (23.3) | 4 (13.3) | 8 (26.7) | 3 (10.0) | 9 (30.0) | 2 (6.7) | ||||
Submucosal invasion | |||||||||||||
Yes | 11 (36.7) | 7 (23.3) | 4 (13.3) | 0.466 | 7 (23.3) | 4 (13.3) | 0.687 | 7 (23.3) | 4 (13.3) | 1 | 9 (30.0) | 2 (6.7) | 0.672 |
No | 19 (63.3) | 9 (30.0) | 10 (33.3) | 14 (46.7) | 5 (16.7) | 13 (43.3) | 6 (20.0) | 13 (43.3) | 6 (20.0) | ||||
Stage | |||||||||||||
pT1a | 19 (63.3) | 9 (30.0) | 10 (33.3) | 0.574 | 14 (46.7) | 5 (16.7) | 0.708 | 13 (43.3) | 6 (20.0) | 0.119 | 13 (43.3) | 6 (20.0) | 0.652 |
pT1b1 | 7 (23.3) | 4 (13.3) | 3 (10.0) | 4 (13.3) | 3 (10.0) | 6 (20.0) | 1 (3.3) | 6 (20.0) | 1 (3.3) | ||||
pT1b2 | 4 (13.3) | 3 (10.0) | 1 (3.3) | 3 (10.0) | 1 (3.3) | 1 (3.3) | 3 (10.0) | 3 (10.0) | 1 (3.3) | ||||
Relapse | |||||||||||||
Yes | 3 (10.0) | 1 (3.3) | 2 (6.7) | 0.586 | 2 (6.7) | 1 (3.3) | 1 | 2 (6.7) | 1 (3.3) | 1 | 0 (0.0) | 3 (10.0) |
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No | 27 (90.0) | 15 (50.0) | 12 (40.0) | 19 (63.3) | 8 (26.7) | 18 (60.0) | 9 (30.0) | 22 (73.3) | 5 (16.7) |
cGAS, cyclic GMP-AMP synthase; IRF3, interferon-regulatory factor 3; STAT6, signal transducer and activator of transcription 6; STING, cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes. Cut-off values for positive cases were defined as a prevalence of >10% of positive tumour cells. Statistical evaluations were conducted using Fisher’s test. The bold values indicates statistical significance.
Kaplan–Meier single-factor analysis using either a >10% or >20% cut-off and associated log-rank tests revealed that cases showing STAT6-positive expression demonstrated a longer progression-free survival time than those with STAT6-negative expression (>10%,
Kaplan–Meier single-factor analysis evaluating progression-free survival in regard to SASP expression in patients with early-stage gastric cancer. Kaplan–Meier single-factor analysis findings were evaluated using log-rank tests. Cut-off values for positive cases were defined as a prevalence of >10% of positive tumour cells. Abbreviations: SASP, senescence-associated secretory phenotype.
Gastric cancers are a genetically and phenotypically heterogeneous group of diseases, as revealed in the Cancer Genome Atlas (TCGA) [
Previous research has focused on cGAS-STING signalling-mediated SASP activity as a regulation network. Beyond an innate immune reaction, SASP is involved in various pathologic processes, including cancer progression, inflammation, autoimmune disease, and aging. Like other reactive mechanisms, SASP signalling is a double-edged sword in regard to tumorigenesis. More specifically, cellular senescence is considered a barrier against transformation, and the eradication of senescent cells is crucial for the clearance of retaliated tissues [
Using ESD specimens, SASP factors (cGAS, STING, IRF3, and STAT6) were found to be highly expressed, and strong SA-β-gal staining was detected in early gastric cancerous cells as well as in high-grade dysplastic lesions. Elevated expression of cGAS, IRF3, and STAT6 was mainly detected in neoplastic lesions, while STING expression occurred frequently in the surrounding non-neoplastic tissue. Our findings demonstrated the prevalent activity of SASP in early neoplastic lesions, suggesting that SASP activity may mainly be a cancer-related event. We also note that STING staining was high in stromal cells and that careful evaluation is therefore necessary. Antibodies for STING staining in FFPE are currently limited. However, the development of more suitable antibodies is expected.
The signalling cascade for SASP is triggered by cGAS activation, which occurs upon the binding of cGAS to DNA; this cascade has been identified to originate from cytosolic DNA or in the formation of micronuclei due to DNA damage [
We detected increased expression of FANCD2, TP53BP1, and RPA2 in early gastric cancer, suggesting that DNA damage and DDR are prevalent in the course of this disease. Accelerated cell proliferation and the resultant expansion of the proliferation zone are generally prevalent in early gastric carcinogenesis. Moreover, we proved that the increased expression of MCM7 in early gastric cancer was consistent with the expression of Ki67, reflecting the proliferation status of the evaluated cells. Interestingly, the expression levels of Ki67 and MCM7 statistically correlated with the expression of FANCD2, TP53BP1, and RPA2, indicating that cell proliferation was closely correlated with DNA damage. The expression of TP53BP1 positively correlated with the expression of cGAS, STING, and IRF3, while the expression of FANCD2 positively correlated with the expression of IRF3 and STAT6, suggesting the major role of DDR in SASP activation in early gastric cancers. In addition, SASP activity was positively associated with DDR, suggesting that SASP may be activated by DNA damage in early gastric cancer. The close correlation between DDR and SASP reinforced the observation that SASP could be associated with neoplastic lesions.
To date, several classification systems for the evaluation of early gastric carcinogenesis have been proposed and implemented clinically; these systems are generally based on morphological changes in gastric epithelia [
Consistent distributions of SASP and DDR expression and
In summary, our investigation suggests that SASP is prevalent in the neoplastic process of the gastric mucosa, and is stimulated by accelerated cell proliferation-induced DDR. Additionally, we reconfirmed that the combination of profound cell proliferation with DDR and SASP expression and
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/
The studies involving human participants were reviewed and approved by the ethics committee of Peking University First Hospital. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements. Written informed consent was not obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.
Concept and design: BZ and TL; experiments, data analysis, and interpretation: LL, YC, and FC; manuscript drafting and editing: LL and BZ; data acquisition and literature search: HL, NM, HZ, SZ, and LN.
This project was supported in part by the Chinese National Natural Science Foundation (No. 81872018), and was also funded in part as a Key Project by the Chinese Ministry of Science and Technology (2017YFC0110205).
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
The Supplementary Material for this article can be found online at:
Kaplan–Meier single-factor analysis of DDR expression, TP53 mutation status, and cell proliferation in reference to progression-free survival in patients with early gastric cancer. Kaplan–Meier single-factor analysis was carried out using log-rank tests. The cut-off values for positive cases were defined as >10% of positive tumour cells. Abbreviations: DDR, DNA damage response.