Identification of NSP3 (SH2D3C) as a Prognostic Biomarker of Tumor Progression and Immune Evasion for Lung Cancer and Evaluation of Organosulfur Compounds from Allium sativum L. as Therapeutic Candidates

Abstract

The novel SH2-containing protein 3 (NSP3) is an oncogenic molecule that has been concomitantly associated with T cell trafficking. However, its oncological role in lung cancer and whether it plays a role in modulating the tumor immune microenvironment is not properly understood. In the present in silico study, we demonstrated that NSP3 (SH2D3C) is associated with advanced stage and poor prognoses of lung cancer cohorts. Genetic alterations of NSP3 (SH2D3C) co-occurred inversely with Epidermal Growth Factor Receptor (EGFR) alterations and elicited its pathological role via modulation of various components of the immune and inflammatory pathways in lung cancer. Our correlation analysis suggested that NSP3 (SH2D3C) promotes tumor immune evasion via dysfunctional T-cell phenotypes and T-cell exclusion mechanisms in lung cancer patients. NSP3 (SH2D3C) demonstrated a high predictive value and association with therapy resistance in lung cancer, hence serving as an attractive target for therapy exploration. We evaluated the in silico drug-likeness and NSP3 (SH2D3C) target efficacy of six organosulfur small molecules from Allium sativum using a molecular docking study. We found that the six organosulfur compounds demonstrated selective cytotoxic potential against cancer cell lines and good predictions for ADMET properties, drug-likeness, and safety profile. E-ajoene, alliin, diallyl sulfide, 2-vinyl-4H-1,3-dithiin, allicin, and S-allyl-cysteine docked well into the NSP3 (SH2D3C)-binding cavity with binding affinities ranging from −3.5~−6.70 Ă and random forest (RF) scores ranging from 4.31~5.26 pKd. In conclusion, our study revealed that NSP3 is an important onco-immunological biomarker encompassing the tumor microenvironment, disease staging and prognosis in lung cancer and could serve as an attractive target for cancer therapy. The organosulfur compounds from A. sativum have molecular properties to efficiently interact with the binding site of NSP3 and are currently under vigorous preclinical study in our laboratory.

Keywords: NSCLC; NSP3 (SH2D3C); dysfunctional T-cell phenotypes; immune infiltrations; in silico study; organosulfur compounds; pharmacokinetics; tumor microenvironment.

Figure 1

Non-structural protein 3 (NSP3; SH2D3C) is associated with advanced stages and poor prognoses of lung cancer cohorts. (A) Box plots showing differential expression profile of NSP3 (SH2D3C) between tumor and adjacent normal tissue across all TCGA cancer types and (B) between tumor and adjacent normal tissues in lung cancer cohort. (C) Violine plots showing differential expression profile of NSP3 (SH2D3C) between normal tissue, tumor, and metastatic lung cancer patients. (D) Kaplan–Meier plot of the overall and progressive-free survival between lung adenocarcinoma patients with low and high expression levels of NSP3 (SH2D3C). (E) Representative immunohistochemistry staining of SH2D3C between lung cancer tumor and pathological free tissue in the Human Protein Atlas (HPA) database. (F) Interactive survival scatter plots and Kaplan–Meier plot of the survival probability between high and low mRNA expression levels in lung cancer cohorts. * p < 0.05; ** p < 0.01; *** p < 0.001.

Figure 2

Genetic alterations of non-structural protein 3 (NSP3; SH2D3C) are associated with poorer prognosis and inversely associated with Epidermal Growth Factor Receptor (EGFR) alterations in lung cancer patients. Genetic alteration profile of NSP3 (SH2D3C) in lung adenocarcinoma. (A) Prevalence and distribution of NSP3 (SH2D3C) genetic alterations in lung adenocarcinoma cohort. (B) Specific mutation types of NSP3 (SH2D3C) in lung adenocarcinoma cohorts. (C) Lollipop plot of NSP3 (SH2D3C) mutation location in lung adenocarcinoma cohort across the cBioPortal for Cancer Genomics dataset. Mutations are color-coded as missense, truncating, and in-frame mutations. (D) Kaplan–Meier plot of the (D) shorter overall survival (E), disease-free survival, and (F) progression-free survival between lung cancer cohorts with genetically altered and cohorts without altered NSP3 (SH2D3C). (G) Heat map of NSP3 (SH2D3C) mutation co-occurrence in lung cancer patients.

Figure 3

Non-structural protein 3 (NSP3; SH2D3C) elicits its pathological role via modulation of various components of the immune and inflammatory pathways in lung cancer. (A) Gene−gene interaction (GGI) and (B) protein−protein interaction (PPI) network of NSP3. (C) The Kyoto Encyclopedia of Genes and Genomes and (D) G.O. biological function enrichments of the NSP3 network proteins. (E) Gene-diseases associated tree plot of NSP3.

Figure 4

Non-structural protein 3 (NSP3; SH2D3C) promotes dysfunctional T-cell phenotypes in lung cancer patients. (A) Scatterplot showing correlations between NSP3 (SH2D3C) expression and infiltration of various immune cells in lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC). (B) Correlation analysis between NSP3 (SH2D3C) expression and cytotoxic T lymphocyte (CTL) levels in lung cancer patients. (C) Kaplan–Meier plot of the overall survival of lung cancer patients with different NSP3 (SH2D3C) and CTL levels. (D) Boxplot showing NSP3 (SH2D3C) expression correlations with infiltration levels of immunosuppressive cells, including cancer-associated fibroblasts (CAFs), M2 macrophages, and regulatory T cells in LUAD patients.

Figure 5

Non-structural protein 3 (NSP3; SH2D3C) methylation and copy number alterations (CNA) are associated with infiltration of immune cells and poorer prognosis of lung cancer cohorts. (A) Heatmap plot of the NSP3 (SH2D3C) expression and T-cell profile in LUAD and LUSC cohorts. (B) Kaplan–Meier plot of the overall survival differences between high and low CNA of NSP3 (SH2D3C) in LUAD and LUSC cohorts. (C) Boxplot showing the association between different somatic copy number alterations and infiltration of various immune cells in LUAD and LUSC. (D) Boxplot showing differential NSP3 (SH2D3C) methylation between lung cancer tumors and adjacent normal tissue. (E) Kaplan–Meier plot of the overall survival differences between lung cancer cohorts with high and low methylation levels of NSP3 (SH2D3C). (F) Kaplan–Meier plot of the overall survival of lung cancer patients with different NSP3 (SH2D3C) methylation status and CTL levels. The “eye” sign represent the hidden view of the CTL, dysfunctional and risk plots of the cohorts.

Figure 6

Non-structural protein 3 (NSP3; SH2D3C) is associated with chemotherapy resistance in NSCLC. (A) Bar plots showing the sensitivity of various clinical drugs between samples with low and high expression levels of SH2D3C. (B) Scatter plot of the efficacy of SH2D3 knockdown (shSH2D3C) in association with the expression of a number gene. The predictivity is defined as the fold change (FC) of shSH2D3C efficacy between samples of high and low expression of the gene. Descriptivity is defined as the FC of gene expression between samples of high and low shSH2D3C efficacy. (C) Bar plot of the comparative biomarker relevance between the SH2D3C and standardized biomarkers.

Figure 7

Blood−brain barrier permeation and in silico cytotoxic properties of some organosulfur compounds in garlic. (A) Blood−brain barrier penetration (BBB) permeation curve of some organosulfur compounds in A. sativum. The BBB permeation ability was measured by the support vector machine (SVM) and LiCABEDS algorithms of the BBB prediction server. (B) In silico cytotoxic properties of some organosulfur compounds in garlic against cancer cell lines. Pa = activity, pi = inactivity.

Figure 8

Molecular docking profile on NSP3 with the organosulfur small molecule from Allium sativum. Two-dimensional (2D) structure and binding surface flip of the ligand−receptor interactions between NSP3 (SH2D3C) and (a) S-allyl-cysteine, (b) E-ajoene, and (c) diallyl sulfide.

Figure 9

Molecular docking profile on novel SH2-containing protein 3 (NSP3) with the organosulfur small molecule from Allium sativum. Three- (3D) and two-dimensional (2D) structure of the ligand−receptor interactions between NSP3 (SH2D3C) and alliin, allicin, and 2-vinyl-4H-1,3-dithiin.