Potential Role of S-Palmitoylation in Cancer Stem Cells of Lung Adenocarcinoma

Abstract

S-palmitoylation, catalyzed by a family of 23 zinc finger Asp-His-His-Cys (DHHC) domain-containing (ZDHHC) protein acyltransferases localized on the cell membrane. However, stemness genes modulated by ZDHHCs in lung adenocarcinoma (LUAD) remain to be defined. Previously, we have constructed a network of cancer stem cell genes, including INCENP, based on mRNA stemness indices (mRNAsi) of LUAD. INCENP has the function of a chromosomal passenger complex locating to centromeres, which is performed by the conserved region of its N-terminal domain. INCENP protein with a deletion of the first non-conserved 26 amino acid sequence failed to target centromeres. However, the exact function of the deleted sequence has not been elucidated. To identify novel cancer stem cell-relevant palmitoylated proteins and responsible ZDHHC enzymes in LUAD, we analyzed multi-omics data obtained from the database of The Cancer Genome Atlas (TCGA), Gene Expression Omnibus (GEO), Clinical Proteomic Tumor Analysis Consortium (CPTAC), and the Human Protein Atlas (HPA). ZDHHC5 is distinguished from the ZDHHC family for being up-regulated in mRNA and protein levels and associated with malignant prognosis. ZDHHC5 was positively associated with INCENP, and the correlation score increased with LUAD stages. CSS-Palm results showed Cys¹⁵ was the S-palmitoylation site of INCENP. Interestingly, Cys¹⁵ locates in the 1-26 aa sequence of INCENP, and is a conserved site across species. As INCENP is a nuclear protein, we predicted that the nuclear localization signal of ZDHHC5 was specific to the importin αβ pathway, and the result of immunofluorescence proves that ZDHHC5 is located in the nucleoplasm, in addition to the plasma membrane. Therefore, our study indicates the S-palmitoylation of INCENP mediated by ZDHHC5 as a potential mechanism of S-palmitoylation to modulate CSCs in LUAD.

Keywords: INCENP; S-palmitoylation; ZDHHC5; cancer stem cell; lung adenocarcinoma; mRNAsi; machine learning.

FIGURE 1

General profiling of ZDHHCs family gene expression and survival analysis in LUAD. (A) The heat map shows the RNA-seq results of ZDHHC mRNA in cases from the LUAD data set of TCGA. The LUAD cases are shown separately by clinical stages. The ZDHHCs coding in green indicate lower expression of the LUAD cases than normal tissues, and the red indicates higher expression. The color of scale bar presents high (red) and low (blue) expression, respectively, and the intensity of color indicates the value of mRNA expression. (B) The differential proteomic expression of ZDHHCs in LUAD cases from the CPTAC database. In the bar plot, the length of the bar represents the normalized expression (Z-value) of ZDHHC in LUAD. The color of the bar presents LUAD (red) and normal lung (blue). (C) Heat map of log₁₀ (HR) illustrating the prognosis of ZDHHCs in LUAD patients from the TCGA database. The scale bar presents high (red) and low (blue) risk, respectively, and the intensity indicates the HR. The bounding box around the tiles represent statistically significant cancer types (HR > 1, P < 0.05). HR, hazard ratio; OS, overall survival; DFS, disease free survival. (D) The Venn diagram shows the mapping results of 3 ZDHHC gene sets. Purple, ZDHHCs that are differentially expressed at the mRNA level; red, ZDHHCs that are differentially expressed at the protein level; green, ZDHHCs with prognostic value. *P < 0.05, **P < 0.01, ***P < 0.001.

FIGURE 2

Expression of ZDHHC5 in LUAD. (A) Fold change of ZDHHC5 differential expression in datasets of lung adenocarcinoma from the GEO and TCGA database. (B) Box plot indicates the differential protein expression of ZDHHC5 in the LUAD sample from CPTAC. Z-value, standard deviations from the median across LUAD samples. **P < 0.01, ***P < 0.001. (C) Distribution of ZDHHC5 expression in LUAD across immune subtypes. (D) Box plot showing mRNA expression of ZDHHC5 in LUAD based on TP53 mutation status. (E) Box plot showing the relative transcription of ZDHHC5 in histological subtypes of LUAD patients. LUAD-not otherwise specified (NOS), LUAD mixed subtype (Mixed), lung solid pattern predominant adenocarcinoma (Solid), lung acinar adenocarcinoma (Acinar), lung micropapillary adenocarcinoma (Micropapillary), lung papillary adenocarcinoma (Papillary). (F) Representative immunohistochemistry results of ZDHHC5 in LUAD. We evaluated histological subtypes to address the heterogeneity of ZDHHC5 in growth patterns of LUAD and normal lung tissues. The histological subtype was classified by a clinical pathologist, and semiquantitative estimations of the different histological patterns present in 5% increments according to the 2015 WHO Classification of Lung Tumors. Enteric adenocarcinoma (Enteric); lepidic adenocarcinoma (Lepidic). (G) Forest plot showing adjusted analysis of ZDHHC5 survival probability on OS in LUAD patients from 17 independent studies. The summarized HR is 1.19, P < 0.05. CI, confidence interval. (H) Kaplan-Meier survival curves of OS and DFS were generated for the comparison of ZDHHC5’s prognostic value in the 3 molecular subtypes of LUAD. The threshold of significance is P < 0.05 in Log-rank test. PP, Proximal-proliferative; PI, proximal-inflammatory; TRU, Terminal respiratory unit.

FIGURE 3

Correlations of ZDHHC5 and INCENP in LUAD. (A) Data for the top 50 positive and negative ZDHHC5 association genes are visualized in heat maps. (B) Venn diagram showing the mapping results of LUAD stemness genes, ZDHHC5 positive and negative expressed genes. (C) The bar plot shows the RNA-seq results of ZDHHC5 and INCENP in A549, HBEC3, and SCLC-21H cell lines from the HPA database. Normalized expression, NX. (D) Correlation analysis of ZDHHC5 and INCENP according to the pathological stages of LUAD cases from TCGA, using Pearson’s correlation tests. (E) The enriched KEGG pathway result clustered by weighted set cover. In the volcano plots, the intensity of color and jitter size indicates the number of the elements in each pathway. (F) GSEA results of cell cycle (hsa04110). NES, normalized enrichment score.

FIGURE 4

Palmitoylation sites of INCENP and its function in cancer stem cell. (A) The palmitoylation sites prediction for INCENP was performed by CCS-Palm 4.0, with the INCENP protein sequence. Cysteine, Cys, C. The color presents high (pink), medium (blue) and low (blue) palmitoylation score, respectively. The significant domains were marked pink (6-41, INCENP_N), and blue (825-881, INCENP ARK-bind). The region surrounding Cys¹⁵ is shown in detail (bottom). (B) The first 26 amino acid sequences of the INCENP across species. (C) PPI networks of LUAD stemness genes having direct interaction with INCENP.

FIGURE 5

ZDHHC5 is the only nuclear ZDHHC family member correlated with INCENP. (A) Results of ZDHHCs nuclear localization signals (NLSs) are shown in the heatmap, calculated by cNLS Mapper. The cNLS Mapper scans the protein sequence with a window size of 16 amino acid residues for monopartite NLSs and 26–28 amino acid residues for bipartite NLSs. (B) Immunofluorescence images (HPA) show the subcellular localization of the nuclear ZDHHC proteins (green) with reference DAPI (blue) for the nucleus in cell lines. (C) Pearson correlation analysis of INCENP and ZDHHCs in the LUAD. The color represents the corresponding correlation value. Red, positive association; green, negative association. |Pearson coefficient| > 0.3 and P < 0.05 is significant. *P < 0.05, **P < 0.01, ***P < 0.001.