Background
The gasdermins family are proposed to be pore-forming effector proteins that cause membrane permeabilization and pyroptosis [
1]. The name gasdermin (gas-dermin) is originally based on the relative unique expression in the upper gastrointestinal tract and dermis [
2]. In humans, the GSDM family contains six members, including GSDMA, GSDMB, GSDMC, GSDMD, GSDME (a.k.a. DFNA5) and PJVK (a.k.a. DFNB59) (Additional file
1: Fig. S1). In mice, homolog of GSDMB is absent but three forms of GSDMA, GSDMA1-3, and four homologs of GSDMC, GSDMC1-4, are present. All GSDM proteins but PJVK share a similar architecture consisting of a functionally important N-terminal pore-forming domain (GSDM-N), a regulatory C-terminal domain (GSDM-C) and a linker between them. The full-length protein before cleavage is considered to be inactive in inducing pyroptosis. In this autoinhibited state, the C-terminal domain binding to the N-terminal effector domain to repress its activity [
3‐
7]. In vitro, actually, the gasdermin-N and -C domains remained bound together following cleavage at inter-domain linker (Additional file
2: Fig. S2) [
8]. A cleavage within the linker region and consequent separation of the two domains is considered to be prerequisite for activation [
5,
9‐
11]. Once cleaved, the cytotoxic N-terminal fragment is liberated and free to form membrane pores through oligomerization and insertion into the plasma membrane, resulting in loss of osmotic homeostasis, cell swelling, and death.
Despite our increasing knowledge of GSDMD, GSDME and GSDMB, the biological functions and the regulation of GSDM expression and activation remain elusive for most GSDMs. A more comprehensive investigation into the function and mechanism of the GSDM family will be beneficial to elucidate GSDM-mediated diseases and consequently development of GSDM-targeting therapeutics.
Methods
Phylogenetic relationship, gene structure, conserved motif analysis
Evolutionary analyses were conducted in MEGA7 [
12]. Gene structure was analyzed by GSDS 2.0 [
13]. Conserved motifs were identified using the online tool MEME 5.3.3 [
14].
Genomic alteration analysis
Analysis of genomic alteration of GSDM members in pan-cancer was performed by Cbioportal (
http://www.cbioportal.org/) [
15,
16]. All types of TCGA Pan-Cancer Atlas available (10,967 samples) were selected for calculation. Mutation including missense, truncating, in frame and fusion gene were examined in the coding sequence of each gene.
Gene expression and survival analysis
TCGA pan-cancer data RNA-Seq (RNA SeqV2 RSEM) and patient survival data were download from Xena browser (
https://xenabrowser.net/datapages/). 33 types of cancer data were obtained, involving ACC, BLCA, BRCA, CESC, CHOL, COAD, DLBC, ESCA, GBM, HNSC, KICH, KIRC, KIRP, LAML, LGG, LIHC, LUAD, LUSC, MESO, OV, PAAD, PCPG, PRAD, READ, SARC, SKCM, STAD, TGCT, THCA, THYM, UCEC, UCS and UVM. Cancer types with no more than 5 normal adjacent tissue as control (15 cancer types) was excluded when analyze the differential expression. Genetical regulation of GSDM gene expression was analyzed by Cbioportal(
http://www.cbioportal.org/) [
15,
16].
Protein–protein interaction analysis
The proteins interacting with GSDM genes were searched from BioGRID 4.3.195 (
https://thebiogrid.org/), followed by visualization by Cytoscape 3.8.1.
Tumor microenvironment analysis
The estimated score was generated by the ssGSEA algorithm and the R script followed as estimateScore (input.ds = "commonGenes.gct", output.ds = "estimateScore.gct").
Immunohistochemistry
A total of 16 human CRC tissue were obtained from patients who had undergone open surgery for cancer resection at the First Affiliated Hospital of Xi'an Jiaotong University (Xi'an, China) between Dec. 2020 and Jan. 2021. Tissues were then formalin-fixed, embedded in paraffin wax and sectioned into 4 μm thick sections. The sections were incubated with primary antibodies against GSDME (1:200, ab215191, Abcam), CD34 (1:800, 14,486-1-AP, Proteintech) overnight at 4˚C, followed by HRP-conjugated secondary goat antibody (ZSGB-Bio). The staining intensity was divided into scores 0, 1, 2 and 3 representing none, weak, moderate, and strong, respectively. The percentage of positive cells was separated into < 5%, 5–25%, 25–50%, 50–75% and ≥ 75% representing 0, 1, 2, 3 and 4 respectively. Immunoreactivity score (IRS) was generated by multiplying the staining intensity score by the percentage of positive cells score. GSDME IRS < 6 was considered as low expression of GSDME, while IRS ≥ 6 was considered as high expression of GSDME.
Cell cultures and lentiviral vectors and transfection
Colon cancer cells HCT116 and SW480 (Shanghai Institute of Cell Biology, Chinese Academy of Sciences) were all routinely cultured in Dulbecco’s modified Eagle’s medium (DMEM) (BI-USA) supplemented with 9% fetal bovine serum (FBS)(LONSERA) at 5% CO2 at 37 °C. The phU6-EGFP-GSDME lentiviral vector and its control vector was constructed and prepared by GeneChem Co., Ltd. All transfections were performed according to the manufacturer's instructions.
Western blot assay
Cells were lysed with RIPA buffer (Beyotime) containing protease inhibitor. Cell lysates were heat denatured, separated by SDS-PAGE, transferred to PVDF membranes (Invitrogen), blocked with 5% skimmed milk. Then membranes were incubated respectively with primary antibodies: GSDME (1:500, ab215191, Abcam), GAPDH (1:5000, 10494-1-AP, Proteintech) overnight at 4℃ and secondary antibodies, goat anti-rabbit IgG (1:5000, bs-0295G, Bioss) for 1 h at room temperature. Immunoblots were visualized using an ECL detection reagent (Minipore).
Wound-healing assays
HCT116 and SW480 cells were cultured in 6-well plates until 95–100%confluent. Then the monolayers were scratched using pipette tips (10 μL). After scratching, cell was washed and maintained in serum-free medium for 72 h. Each experiment was performed in triplicate.
Transwell assays
Cell migration and invasion were measured using Boyden chambers in 24-transwell plates (8 μm pores, Corning). In both assays, the lower chambers were filled with 600 μL DMEM medium containing 10% FBS. In the migration assay, 2.5 × 104 cells were seeded in upper chamber. In invasion assay, 5 × 104 cells were seeded in upper chamber pre-coated with 60 μL of Matrigel (BD). The cells were incubated at 37 °C, 5% CO2 for 48 h. After incubation, non-migratory cells were removed by cotton swab. Then the membranes were fixed with 4% paraformaldehyde and stained with 1% crystal violet. The number of cells was counted in five randomly selected fields. Each experiment was performed in triplicate.
Statistics analysis
The Wilcoxon Signed-rank Test was used for analyzing the differential expression of GSDM gene between tumor and normal tissue. Kruskal–Wallis rank sum test was used for comparing GSDM gene expression among immune subtypes. Pearson's correlation tests were used for analyzing the correlation among GSDM gene members and the correlation between GSDM gene expression and z scores for cell sensitivity data. Spearman's Rank-Order Correlation was used for analyzing association between gene expression and Estimate/Stemness scores. The Kaplan–Meier method with log-rank test were used for comparing survival. Cox regression was used for analyzing risk factors on survival. P value < 0.05 indicated statistical significance. Besides online analysis, all statistics were calculated by R Project (Version 4.0.3).
Discussion
Since its first discovery more than two decades ago, a great amount of effort has been made to investigate the biofunction of GSDM family genes. Originally, GSDM genes were identified as candidate causative genes of hair loss and hearing loss [
27‐
30]. The real breakthrough was that GSDMD was identified as the key executor of inflammatory cell death known as pyroptosis [
5,
9,
31,
32], a lytic type of cell death associated with inflammation. Because of their similar architecture, we tend to believe all GSDM genes have the ability somehow to form pores in plasm or mitochondrial membrane and to trigger cell death following N-terminal activation. Therefore, most studies have focused on which site of GSDM gene has been cleaved by which enzymes, the caspase or other, thus activating the specific GSDM gene, forming pores in cells subsequently, triggering lytic cell death. Later, GSDME and GSDMB have been uncovered to play a critical role in inducing pyroptosis [
11,
27], further reinforcing the idea that GSDM genes have the pore-forming and cell lysis ability. To date, however, there is little known about GSDMA, GSDMC and PJVK, although their N-terminal moieties generated by genetical engineering in vitro showed pore-forming activity that binds to membrane inner leaflet lipids and causes cell membrane permeabilization and pyroptosis [
8,
33]. In addition, GSDM genes have been implicated in regulation of cancer behaviors, but whether it suppresses cancer or promotes cancer is controversial [
34‐
37].
In this pan-cancer study, we found that (1) genome of GSDM genes were extensively altered in cancer and the corresponding patients showed survival disadvantage; (2) the expression of GSDM genes was altered in cancer due to genetic alteration and epigenetic modification of GSDM genome, and it affected patient survival; (3) GSDM family members were involved in cancer-related pathways in varying degrees and had a role in drug sensitivity; (4) the expression of GSDM genes associated with immune subtypes, TME and cell stemness of cancer. Our finding indicated that GSDM genes had an extensive involvement in cancer, more efforts should be made to elucidate the detailed mechanisms. However, we also discovered an intra- and inter-cancer heterogeneity regarding the corresponding genes. We suggest each GSDM gene be studied as an entity in each type of cancer.
Other than triggering pyroptosis, we suspected GSDM genes might play other roles in regulation of cancer cell behaviors. Though our analysis, we found that GSDMA, GSDMB, GSDMD and GSDME might be involved in colorectal cancer cell invasion and metastasis, and the former three might played as negative regulators while the last one played as a positive regulator. The expression of GSDME strongly positive correlation with stromal cells infiltration and the expression of EC markers in solid tissue of CRC. In CCLE, we found the expression of GSDME was significantly positively correlated with the expression of VEGFB and VEGFC in CRC cell lines. Thus, we suspected there was a crosstalk between GSDME and VEGF within the cancer cell and another crosstalk between cancer cell and TME, which was considered to play an important role in cancer progression, metastasis as well as drug resistance [
38‐
42]. Also, our experiment verified that overexpression GSDME promoted the migration and invasion of SW480 and HCT116 cell lines and the positive correlation between GSDME expression and MVD. Upregulated GSDME by reversal of epigenetic silencing and facilitated the occurrence of pyroptosis was considered as an important pyroptosis-based cancer chemotherapy strategy [
43], but we suggest more attention should be paid to the oncogenic role of GSMDE when being upregulated.
There were some limitations in our study. Different online databases usage might cause background heterogeneity. Besides, we didn’t carry out enough experiments to verify our findings. Next, more studies in vitro and in vivo will be carried out to verify the biological information results.
Conclusion
GSDM family genes might play important roles in cancer. We suggest more efforts be made to investigate the GSDM family and each GSDM gene be studied as an entity in each type of cancer. While studying pyroptosis, other roles GSDM genes might play should not be ignored. And, GSDMA, GSDMB and GSDMD exhibited a lot of differences, even opposite aspects to GSDME, and we hope this might provide some reference for the future research.
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