Identification of hub genes and compounds controlling ovarian cancer stem cell characteristics via stemness indices analysis

Abstract

Background: Ovarian cancer (OC) is the most lethal gynecological malignancy. It has been reported that cancer stem cells (CSCs) play a crucial role in disseminated metastases in abdominal cavity and chemotherapy resistance of high-grade serous OC. However, the overall gene expression features of OC stem cells have not been clarified.

Methods: Expression datasets of 379 OC samples and 88 normal tissues were downloaded from The Cancer Genome Atlas (TCGA) and the Genotype Tissue Expression (GTEx) project. Differentially expressed genes (DEGs) were screened using the "limma" package in R software. Among the DEGs, modules and hub genes that were highly related to messenger RNA expression-based stemness index (mRNAsi) and epigenetically regulated mRNAsi indices were identified via weighted gene co-expression network analysis (WGCNA). These hub genes were considered to be associated with OC stem cells. The Gene Ontology (GO) project and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was used to identify the main biological processes that hub genes participated in. Finally, Connectivity Map (CMap) was used to predict compounds that disturb the hub genes.

Results: We identified 2,253 DEGs; of these, 31 had a significantly positive correlation to mRNAsi indices and were upregulated in OC, while 41 of them had a significantly negative correlation with mRNAsi indices and were downregulated in OC. Correlation analysis indicated that hub genes from the same module composed a dense interaction network. GO and KEGG enrichment analysis demonstrated that hub genes primarily play roles in cell division and proliferation. Moreover, the compounds that may disturb hub genes were identified. Of these, 11 compounds, including MS-275, DL-thiorphan, and GW-8510, which have never been studied in OC stem cells, were screened as underlying treatments targeting OC stem cells.

Conclusions: Altogether, 72 hub genes that were closely linked to OC stem cell characteristics were found to mainly participate in cell division and proliferation. Moreover, compounds that disturb these hub gens were identified and can be considered underlying targets for inhibiting OC stem cells.

Keywords: Ovarian cancer (OC); cancer cell stemness; connectivity map (CMap); mRNAsi; weighted gene co-expression network analysis (WGCNA).

Figure 1

Differentially expressed genes (DEGs) in OC and normal samples. (A) A volcano plot depicting the DEGs. The green points indicate the significantly downregulated genes and the red points indicate the significantly upregulated genes. (B) A heatmap illustrating the expression levels of the top 20 ranked upregulated and top 20 ranked downregulated genes in OC and normal samples. OC, ovarian cancer.

Figure 2

Identification of hub gene modules associated with the mRNAsi of OC. (A) Plots showing the scale free topology fitting index. (B) Plots showing the mean connectivity. (C) Hierarchical clustering tree bases on the topological overlap dissimilarity (1-TOM). The tree branches of the hierarchical clustering are colored by module membership. (D) A heatmap depicting the correlations between the gene modules and clinical significances, including both mRNAsi and EREG-mRNAsi. The corresponding P values are also annotated. (E) Scatter plots representing the correlation between the brown module membership and the gene significance for mRNAsi. (F) Scatter plots representing the correlation between the green module membership and the gene significance for mRNAsi. mRNAsi, messenger RNA expression-based stemness index; OC, ovarian cancer; EREG-mRNAsi, epigenetic regulation-based index.

Figure 3

Box plots depicting the expression levels of hub genes derived from (A) the brown module. and (B) the green module between OC and normal samples. The red box indicates the OC tissue and the blue box indicates normal tissues. ***, P<0.001. OC, ovarian cancer.

Figure 4

Correlation between hub genes derived from (A) the brown module and (B) the green module. The upper part of these two figures shows the level of confidence that the two proteins are functionally associated, which is indicated by the intensity of the color while the lower part annotates the corresponding correlation value (Pearson’s correlation coefficient).

Figure 5

PPI networks of hub genes. PPI networks show PPI between hub genes derived from (A) the brown module and (C) the green module. The minimum required interaction score threshold was set as 0.4, and disconnected nodes in the network are hidden. Bar plots showing the nodes of hub genes derived from (B) the brown module and (D) the green module. PPI, protein-protein interaction.

Figure 6

Functional analysis of the hub genes. (A) A bubble plot showing the top 10 ranked terms in groups of biological process (BP), cellular component (CC), and molecular function (MF). (B) A bubble plot showing the significantly enriched KEGG pathways of the hub genes. KEGG, Kyoto Encyclopedia of Genes and Genomes.

Figure 7

The 3D conformers of the selected compounds that were obtained from CMap analysis. (A) MS-275, (B) DL-thiorphan, (C) piperlongumine, (D) thioguanosine, (E) GW-8510, (F) trifluridine, (G) alsterpaullone, (H) clomipramine, (I) phenoxybenzamine, (J) sulconazole, (K) pyrvinium. CMap, connectivity map.