Supplementary Figures and Tables - Figure S1. RNF40 somatic copy number alterations in a cohort of 579 HGSOC cases from The Cancer Genome Atlas (TCGA). Heterozygous loss of RNF40 is present in 36% of HGSOCs. Figure S2A. H2Bub1 stain intensity dot plot. Figure S2B. Distribution of H2Bub1 immunoreactivity (% positive cells). Figure S2C. Immunohistochemical analysis of Total H2B and H2Bub1 levels in invasive high-grade serious ovarian cancer. Figure S3. Stable RNF20 and H2Bub1 depletion escalates oncogenic behavior in FTSEC cell lines. Figure S4. Transient RNF20 and H2Bub1 depletion escalates oncogenic behavior in FTSECs. Figure S5. RNF20-depletion changes chromatin accessibility and redistributes open chromatin. Figure S6. RNF20-depletion changes chromatin accessibility and alters gene expression of immune signaling pathways in FTSEC cells. Figure S7. RNF20 and H2Bub1 deficiency alters the levels of cytokines. Figure S8. Representative images for RNA in situ hybridization of IL6 expression in FFPE tumor and normal tissue sections. Supplementary Table 1. Antibody information and staining conditions for immunohistochemistry (IHC).
Funding
National Cancer Institute
NIH
European Research Council
American Cancer Society Early Detection Professorship
ARTICLE ABSTRACT
Recent insights supporting the fallopian tube epithelium (FTE) and serous tubal intraepithelial carcinomas (STIC) as the tissue of origin and the precursor lesion, respectively, for the majority of high-grade serous ovarian carcinomas (HGSOC) provide the necessary context to study the mechanisms that drive the development and progression of HGSOC. Here, we investigate the role of the E3 ubiquitin ligase RNF20 and histone H2B monoubiquitylation (H2Bub1) in serous tumorigenesis and report that heterozygous loss of RNF20 defines the majority of HGSOC tumors. At the protein level, H2Bub1 was lost or downregulated in a large proportion of STIC and invasive HGSOC tumors, implicating RNF20/H2Bub1 loss as an early event in the development of serous ovarian carcinoma. Knockdown of RNF20, with concomitant loss of H2Bub1, was sufficient to enhance cell migration and clonogenic growth of FTE cells. To investigate the mechanisms underlying these effects, we performed ATAC-seq and RNA-seq in RNF20 knockdown FTE cell lines. Loss of RNF20 and H2Bub1 was associated with a more open chromatin conformation, leading to upregulation of immune signaling pathways, including IL6. IL6 was one of the key cytokines significantly upregulated in RNF20- and H2Bub1-depleted FTE cells and imparted upon these cells an enhanced migratory phenotype. These studies provide mechanistic insight into the observed oncogenic phenotypes triggered by the early loss of H2Bub1.
Loss of RNF20 and H2Bub1 contributes to transformation of the fallopian tube epithelium and plays a role in the initiation and progression of high-grade serous ovarian cancer.
