A disintegrin and metalloproteinase domain 10 expression inhibition by the small molecules adenosine, cordycepin and N6, N6-dimethyladenosine and immune regulation in malignant cancers

Abstract

A disintegrin and metalloproteinase domain 10 (ADAM10), a member of the ADAM family, is a cellular surface protein with potential adhesion and protease/convertase functions. The expression regulations in cancers by natural products [adenosine (AD) and its analogs, cordycepin (CD), and N6, N6-dimethyladenosine (m⁶ ₂A)], and immune regulation are unclear. As results, AD, CD, and m⁶ ₂A inhibited ADAM10 expression in various cancer cell lines, indicating their roles in anti-cancer agents. Further molecular docking with ADAM10 protein found the binding energies of all docking groups were <-7 kcal/mol for all small-molecules (AD, CD and m⁶ ₂A), suggesting very good binding activities. In addition, analysis of the immunomodulatory roles in cancer showed that ADAM10 was negatively correlated with immunomodulatory genes such as CCL27, CCL14, CCL25, CXCR5, HLA-B, HLA-DOB1, LAG3, TNFRSF18, and TNFRSF4 in bladder urothelial carcinoma, thymoma, breast invasive carcinoma, TGCT, kidney renal papillary cell carcinoma, SKCM and thyroid carcinoma, indicating the immune-promoting roles for ADAM10. LAG3 mRNA levels were reduced by both AD and CD in vivo. ADAM10 is also negatively associated with tumor immunosuppression and interrelated with the immune infiltration of tumors. Overall, the present study determined ADAM10 expression by AD, CD and m⁶ ₂A, and in AD or CD/ADAM10/LAG3 signaling in cancers, and suggested a potential method for immunotherapy of cancers by targeting ADAM10 using the small molecules AD, CD and m⁶ ₂A.

Keywords: ADAM10; CD; adenosine; expression; immunotherapy; m62A; pan-cancer.

Figure 1

AD inhibits ADAM10 expression in various cancer cells. (A) AD inhibited expression of ADAM10 in both protein showing in upper panel and mRNA showing in lower panel in the H1975 lung cancer cells. (B) AD inhibited expression of ADAM10 in both protein showing in upper panel and mRNA showing in lower panel in the A549 lung cancer cells. (C) AD inhibited expression of ADAM10 in both protein showing in upper panel and mRNA showing in lower panel in the BT549 breast cancer cells. (D) Quantitative results for ADAM10 protein levels. (E) Quantitative results for ADAM10 mRNA levels. AD, adenosine; ADAM10, a disintegrin and metalloproteinase domain 10. *P <0.05; **P < 0.005; ****P < 0.0001.

Figure 2

Cordycepin (CD) inhibits ADAM10 expressions in various cancer cells. (A) CD inhibited expression of ADAM10 in both protein showing in upper panel and mRNA showing in lower panel in the H1975 lung cancer cell line. (B) CD inhibited expression of ADAM10 in protein showing in upper panel and mRNA showing in lower panel in the A549 lung cancer cell line. (C) CD inhibited expression of ADAM10 in protein showing in upper panel and mRNA showing in lower panel in the BT549 breast cancer cell line. (D) Quantitative results for ADAM10 protein levels. (E) Quantitative results for ADAM10 mRNA levels. CD, cordycepin; ADAM10, a disintegrin and metalloproteinase domain 10. *P <0.05; ****P < 0.0001.

Figure 3

m⁶ ₂A inhibits ADAM10 expressions in various cancer cell lines. (A) m⁶ ₂A inhibited expression of ADAM10 in both protein showing in upper panel and mRNA showing in lower panel in the H1975 lung cancer cell line. (B) m⁶ ₂A inhibited expression of ADAM10 in both protein showing in upper panel and mRNA showing in lower panel in the A549 lung cancer cell line. (C) m⁶ ₂A inhibited expression of ADAM10 in both protein showing in upper panel and mRNA showing in lower panel in the BT549 breast cancer cell line. (D) Quantitative results for ADAM10 protein levels. (E) Quantitative results for ADAM10 mRNA levels. m⁶ ₂A, N6, N6-dimethyladenosine; ADAM10, a disintegrin and metalloproteinase domain 10. ****P < 0.0001.

Figure 4

ADAM10 stability by AD and CD. (A) ADAM10 stability by AD treatments. Right panel, quantitative results. (B) ADAM10 stability by CD treatments. Right panel, quantitative results. CHX (20µg/ml) was used to treat A549 lung cancer cell lines or BT549 breast cancer cell lines with or without indicated AD or CD treatments, and western blot was performed. β-actin was used as an internal control. CHX, cycloheximide.

Figure 5

Molecular docking for ADAM10 (6bdz) and AD, CD, and m⁶ ₂A binding sites. (A) Molecular docking of AD with human ADAM10 protein (6bdz) and active site residues and (B) force of attractions involved in docking; (C) Molecular docking of CD with human ADAM10 protein (6bdz) and active site residues and (D) force of attractions involved in docking; (E) Molecular docking of AD with human ADAM10 protein (6bdz) and active site residues and (F) force of attractions involved in docking.

Figure 6

Bioinformatic analysis for the immunoregulatory actions of ADAM10 in various cancers. (A) Correlations between ADAM10 and 150 genes of Immunoregulation. There are 41 chemokines, 18 receptors, 21 MHCs, 24 immunoinhibitors, and 46 immunostimulators. (B) Correlations between ADAM10 and 60 genes of immune checkpoint pathways. (C) AD inhibits LAG3 expression in H1975 cells. Up panel indicates semi-quantitative RT-PCR, bottom panel indicates the quantitative results from up panel. (D) CD inhibits LAG3 expression in BT549 cells. Up panel indicates semi-quantitative RT-PCR, bottom panel indicates the quantitative results from up panel. *P <0.05. ADAM10, a disintegrin and metalloproteinase domain 10. For immunomodulatory genetic analysis, the pan-cancer data-set The Cancer Genome Atlas (TCGA) TARGET Genotype-Tissue Expression (GTEx) (PANCAN) was downloaded from University of California Santa Cruz (UCSC; https://xenabrowser.net/).

Figure 7

Pearson’s correlations of the correlation between ADAM10 expression and tumor-related immune cells in various cancers calculated using different methods. (A) Correlation between ADAM10 and six tumor-associated immune cells calculated using Tumor Immune Estimation Resource. (B) Correlations between ADAM10 and six tumor-related immune cells calculated using deconvo_ips. (C) Correlations between ADAM10 and 22 tumor-associated immune cells calculated using deconvo CIBERSOR. *P <0.05; **P < 0.005; ***P <0.001; ****P < 0.0001. ADAM10, a disintegrin and metalloproteinase domain 10.

Figure 8

Association between ADAM10 expression and immune infiltration score in multiple types of cancer. For immune infiltration analysis, the pan-cancer data-set TCGA TARGET Genotype-Tissue Expression (GTEx) was also downloaded from UCSC. ADAM10 expression and immune infiltration score in multiple types of cancer are presented in the indicated different panels.