Abstract
Deregulation of the transforming growth factor-β (TGFβ) signaling pathway in epithelial ovarian cancer has been reported, but the precise mechanism underlying disrupted TGFβ signaling in the disease remains unclear. We performed chromatin immunoprecipitation followed by sequencing (ChIP-seq) to investigate genome-wide screening of TGFβ-induced SMAD4 binding in epithelial ovarian cancer. Following TGFβ stimulation of the A2780 epithelial ovarian cancer cell line, we identified 2,362 SMAD4 binding loci and 318 differentially expressed SMAD4 target genes. Comprehensive examination of SMAD4-bound loci, revealed four distinct binding patterns: 1) Basal; 2) Shift; 3) Stimulated Only; 4) Unstimulated Only. TGFβ stimulated SMAD4-bound loci were primarily classified as either Stimulated only (74%) or Shift (25%), indicating that TGFβ-stimulation alters SMAD4 binding patterns in epithelial ovarian cancer cells. Furthermore, based on gene regulatory network analysis, we determined that the TGFβ-induced, SMAD4-dependent regulatory network was strikingly different in ovarian cancer compared to normal cells. Importantly, the TGFβ/SMAD4 target genes identified in the A2780 epithelial ovarian cancer cell line were predictive of patient survival, based on in silico mining of publically available patient data bases. In conclusion, our data highlight the utility of next generation sequencing technology to identify genome-wide SMAD4 target genes in epithelial ovarian cancer and link aberrant TGFβ/SMAD signaling to ovarian tumorigenesis. Furthermore, the identified SMAD4 binding loci, combined with gene expression profiling and in silico data mining of patient cohorts, may provide a powerful approach to determine potential gene signatures with biological and future translational research in ovarian and other cancers.
Highlights
The transforming growth factor-b (TGFb) signaling pathway plays an important role in controlling proliferation, differentiation, and other cellular processes including the growth of ovarian surface epithelial cell (OSE) [1,2]
In order to further elucidate the details of the underlying mechanisms, we used ChIPseq technology to identify the genomic locations bound by SMAD4 in A2780 cells before and after TGFb stimulation
In order to determine the significance of each percentile, a set of randomly simulated reads is used as a background to estimate the false discovery rate (FDR)
Summary
The transforming growth factor-b (TGFb) signaling pathway plays an important role in controlling proliferation, differentiation, and other cellular processes including the growth of ovarian surface epithelial cell (OSE) [1,2]. The functional receptor complex regulates the activation of downstream Smad and non Smad pathways [8]. Activated R-Smads form heteromeric complexes with the common partner Smad (coSmad; SMAD4 in mammals) and translocate into the nucleus [6]. As the affinity of the activated Smad complex for the Smad-binding element is insufficient to support association with endogenous promoters of target genes, Smad complexes must associate with other DNA binding transcription factors to regulate expression [7]. Numerous studies have shown that various families of transcription factors, such as the forkhead, homeobox, zinc finger, LEF1, Ets, and basic helix–loop–helix (bHLH) families, can serve as SMAD4 partner proteins to achieve high affinity and selectivity for target promoters with the appropriate binding elements [10,11,12,13,14]
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