Multi-omics profiling reveals key signaling pathways in ovarian cancer controlled by STAT3

Abstract

Inhibiting STAT3 signaling reduces tumor progression, metastasis and chemoresistance, however the precise molecular mechanism has not been fully delineated in ovarian cancer.

Methods: In this study, we generated STAT3 knockout (KO) ovarian cancer cell lines. Effects of STAT3 KO on cell proliferation, migration and spheroid formation were assessed in vitro and effects on in vivo tumor growth were tested using several tumor xenograft models. We used multi-omic genome-wide profiling to identify multi-level (Bru-Seq, RNA-Seq, and MS Proteomic) expression signatures of STAT3 KO ovarian cancer cells.

Results: We observed that deletion of STAT3 blocked cell proliferation and migration in vitro and suppressed tumor growth in mice. Deletion of STAT3 transcriptionally suppressed key genes involved in EMT, cell cycle progression, E2F signaling, and altered stemness markers. Notably, KO of STAT3 resulted in modulation of the expression of other STAT family members.

Conclusion: Our study presents a rich, multi-faceted summary of the molecular mechanisms impacted by STAT3 deletion and provides new insight for STAT3's potential as a therapeutic target in ovarian cancer.

Keywords: CRISPR-Cas9; Multi-omic genome-wide analysis; Ovarian cancer; STAT3; STAT3 knockout.

Figure 1

Deletion of STAT3 reduces cell proliferation, migration, and spheroid formation of ovarian cancer cells in vitro. (A). The proliferation rate of SKOV3, OVCAR3, OVCAR8 and HEY WT/ STAT3 KO cells was evaluated by a cell proliferation assay. The graph displays the cell numbers versus time in h. (B). Migration capability of WT and STAT3 KO cells was determined in a wound-healing assay. The panels on the left show the wound at 0 h after the scratch and the right panels show the wound after 24 h. Scale bar, 100 μm. A bar diagram with statistical analysis is provided in Supplemental Figure S2A. (C). Spheroid formation capability of WT and STAT3 KO cells was determined in a 3D spheroid assay. Spheroid growth was imaged at Days 2, 4 and 6. Scale bar, 100 μm. Luminescence representing cell viability of the same experiment was measured using the CellTiter-Glo® 3D Cell Viability Assay and is presented with statistical analysis in Supplemental Figure S2B.

Figure 2

STAT3 KO causes tumor growth inhibition in mouse xenograft models of ovarian cancer. (A-D). Growth curves of tumors in nude mice (n = 5) injected with SKOV3 (A), OVCAR3 (B), OVCAR8 (C) and HEY (D) WT/ STAT3 KO cells. (E) H&E staining of SKOV3 WT/ STAT3 KO xenograft tumor tissue sections. The bottom panels show a higher magnification of the boxed area in the upper panel. Overall histologic tumor size and cellular density of the STAT3 KO specimen was markedly reduced in comparison to the WT specimen histologically. The overall appearance of the STAT3 KO specimen was that of collapse, with fewer tumor cells and more stroma than that of the WT specimen. Cellular features between the two specimens were also observably different. Relatively less anisocytosis and anisokaryosis, with smaller cells, many of which contained more dense cytoplasm and fewer abnormal nuclear features were observed in STAT3 KO specimen. Scale bar, 100 μm. (F) Growth curves of tumors in huNOG mice injected with SKOV3 WT/ STAT3 KO cells (n = 2). (G) NSG mice (n = 5) were co-injected with SKOV3 WT/ STAT3 KO cells, with or without ovarian cancer patient-derived mesenchymal stem cells (MSC). Statistical significance was calculated using Student's t-test. Error bars indicate mean ± SEM (standard error of mean). *p < 0.05, **p < 0.01.

Figure 3

RNA and protein expression profiles of SKOV3 STAT3 parental and KO cells. (A). Differential expression of genes as a result of STAT3 KO across different platforms. (B). A total of 22 genes were commonly up (8 genes) or down (14 genes) regulated across all three profiling platforms. (C). Using DAVID, two Gene Ontology categories “Cell Junction Organization” and “Positive Response to Cell Migration” were commonly enriched for genes differentially expressed in all 3 platforms. (D) Heatmap showing log2 fold changes of 116 genes belonging to the Positive Regulation of Cell Migration gene set that were differentially expressed in at least one platform. (E) Overlap of GSEA enriched gene sets for SKOV3 STAT3 KO. Up-regulated genes included KEGG Antigen Processing and Presentation, and hallmark Interferon Gamma Response. Down-regulated genes included 2 hallmark gene sets: G2/M Checkpoint, and E2F Targets. (F) GSEA enrichment plots for each enriched gene set and platform. NES = normalized enrichment score; FDR= false discovery rate. Differential expression thresholds used per platform are described in methods.

Figure 4

Common genes and pathways altered across three ovarian cancer cell lines in response to STAT3 deletion. (A) Transcriptional profile comparison between parental ovarian cell lines shows a high degree of similarity. (B) STAT3 KO RNA-Seq differential expression across three ovarian cancer cell lines. (C) 19 differentially expressed genes were observed across all 3 cell lines in response to STAT3 KO. (D) Using DAVID, differentially expressed genes were commonly enriched in three Gene Ontology categories. FDR-adjusted p-values for “GO:0030198 Extracellular Matrix Organization” in SKOV3, OVCAR3 and OVCAR8 are 1.51 × 10^-8, 0.0597 and 8.3 × 10^-6, respectively. FDR-adjusted p-values for “GO:0043062 Extracellular Structure Organization” in SKOV3, OVCAR3 and OVCAR8 are 1.72 × 10^-8, 0.0634 and 4.9 × 10^-8, respectively. FDR-adjusted p-values for “GO:0009615 Response to Virus” in SKOV3, OVCAR3 and OVCAR8 are 0.0245, 0.0930, and 0.0363, respectively. (E) The E2F Targets hallmark gene set was significantly enriched for down-regulated genes using GSEA (FDR adjusted p-value < 0.1) in SKOV3 and OVCAR3 cell lines. (F) Kaplan-Meier survival plots of 7 genes have significant associations with patient survival analyzed from TCGA.

Figure 5

STAT3 KO suppresses epithelial-mesenchymal transition. (A). Log2 fold change profile of nascent RNA encoding EMT-regulated genes in SKOV3 STAT3 KO samples. Genes colored red were annotated as pertaining to a mesenchymal phenotype (n=311), while genes colored blue were annotated as pertaining to an epithelial phenotype (n=284) in cancer cells. 14 epithelial- or mesenchymal-related annotated gene sets obtained from the Molecular Signatures Database (MSigDB, GSEA - Broad Institute) were used to generate a list of 747 genes and corresponding epithelial/mesynchemal annotations. Gene list is provided in Supplemental Table S7. Boxplot at bottom left represents log2 fold change average of genes regulating epithelial and mesenchymal phenotypes in SKOV3 STAT3 KO/WT. *** indicate a p-value < 0.001, unpaired Student's t-test, two-tailed p-value. (B). EMT markers and EMT-TFs governing EMT progression are downregulated in SKOV3 STAT3 knockout cells at the RNA and protein levels.

Figure 6

STAT3 KO downregulates expression of genes involved in the G2/M phase. (A). Cell cycle profiling revealed G2/M enrichment in STAT3 KO cells. Cells were fixed, stained with propidium iodide, and DNA content was analysed by flow cytometry. Statistical analysis is provided in Supplemental Table S8. (B) Key cell cycle mediators coding genes were mostly down regulated in SKOV3 STAT3 KO cells compared to WT cells. Colors represented log2 fold change differences between STAT3 KO cell lines and parental cell lines. The impact of STAT3 KO varied greatly in the magnitude of differential expression between Bru-seq and RNA-seq, two scale bars are used. (C). Key cell cycle mediators were suppressed in STAT3 KO cells. Protein expression levels were determined by Western blot.

Figure 7

STAT3 KO alters expression of STAT family members and stemness-like markers. ( A). STAT1 and STAT2 nascent RNA are upregulated in SKOV3 STAT3 KO cells as determined by Bru-Seq analysis. (B). Changes in protein expression of STAT family members in WT/ STAT3 KO cells. (C). RT-PCR of STAT5A and STAT5B expression in STAT3 KO cells compared to control cells. (D). ALDH1A1 nascent RNA is upregulated and CD44 is downregulated in SKOV3 STAT3 KO cells. (E). Protein expression of ALDH1A1, ALDH1A3 and CD44 in WT/ STAT3 KO cells. (F). Expression of STAT3, ALDH1A1 and CD44 in nude xenograft tumors from ovarian cancer cells with STAT3 deletion. (G). Protein expression of STAT3, ALDH1A1 and CD44 upon STAT3 knock-down. SKOV3 cells were treated with 30 nM STAT3 siRNA for 72 h and lysed for Western blot. Gene map is from RefSeq Genes (UCSC genome browser, http://genome.ucsc.edu/) and RPKM directionality indicates strand. RPKM=reads per kilobase of transcript per million mapped reads.

Figure 8

50 significant STAT3 co-expression gene sets in common between 4 diseases. STAT3 co-expression modelled using Gene Set Enrichment Analysis (GSEA) to identify gene sets enriched for genes correlated with STAT3 expression in ovarian (OV), glioblastoma and lower grade glioma (GBMLGG), uveal melanoma (UVM), and kidney renal clear cell carcinoma (KIRC) TCGA cohorts. A full list of gene sets is provided in Supplemental Figure S9B.