The E3 ubiquitin ligase RNF40 suppresses apoptosis in colorectal cancer cells

Abstract

Background Colorectal cancer (CRC) is the fourth leading cause of cancer-related deaths worldwide, and deciphering underlying molecular mechanism is essential. The loss of monoubiquitinated histone H2B (H2Bub1) was correlated with poor prognosis of CRC patients and, accordingly, H2Bub1 was suggested as a tumor-suppressive mark. Surprisingly, our previous work revealed that the H2B ubiquitin ligase RING finger protein 40 (RNF40) might exert tumor-promoting functions. Here, we investigated the effect of RNF40 loss on tumorigenic features of CRC cells and their survival in vitro. Methods We evaluated the effects of RNF40 depletion in several human CRC cell lines in vitro. To evaluate cell cycle progression, cells were stained with propidium iodide and analyzed by flow cytometry. In addition, to assess apoptosis rates, caspase 3/7 activity was assessed in a Celigo® S-based measurement and, additionally, an Annexin V assay was performed. Genomic occupancy of H2Bub1, H3K79me3, and H3K27ac was determined by chromatin immunoprecipitation. Transcriptome-wide effects of RNF40 loss were evaluated based on mRNA-seq results, qRT-PCR, and Western blot. To rescue apoptosis-related effects, cells were treated with Z-VAD-FMK. Results Human CRC cell lines displayed decreased cell numbers in vitro after RNF40 depletion. While the differences in confluence were not mediated by changes in cell cycle progression, we discovered highly increased apoptosis rates after RNF40 knockdown due to elevated caspase 3/7 activity. This effect can be explained by reduced mRNA levels of anti-apoptotic and upregulation of pro-apoptotic BCL2 family members. Moreover, the direct occupancy of the RNF40-mediated H2B monoubiquitination was observed in the transcribed region of anti-apoptotic genes. Caspase inhibition by Z-VAD-FMK treatment rescued apoptosis in RNF40-depleted cells. However, knockdown cells still displayed decreased tumorigenic features despite the absence of apoptosis. Conclusions Our findings reveal that RNF40 is essential for maintaining tumorigenic features of CRC cells in vitro by controlling the expression of genes encoding central apoptotic regulators. Electronic supplementary material The online version of this article (10.1186/s13148-019-0698-x) contains supplementary material, which is available to authorized users.

RNF40-dependent epigenetic regulation of actin cytoskeletal dynamics is required for HER2-driven mammary tumorigenesis

“…3B). Consistent with our previous findings in colorectal cancer (19), Gene Set Enrichment Analysis (GSEA) identified a significant enrichment for a gene signature associated with hallmarks of apoptosis, potentially explaining the reduced oncogenic properties (Fig. 3C and S3B).…”

“…In addition to the central role of actin cytoskeleton dynamics in controlling cellular migration, the ROCK and focal adhesion kinase signaling pathway also has a critical function in suppressing apoptosis (37,51). While we previously identified a role for RNF40 in suppressing apoptosis in colorectal cancer cells via expression of anti-apoptotic members of the BCL2 family of proteins (19), our current results suggest that RNF40 suppresses programmed cell death in HER2-positive BC in a distinct manner via maintenance of ROCK-dependent focal adhesion kinase signaling. Indeed, focal adhesion structures decreased in size with a concomitant decrease in FAK kinase activity upon either RNF40 depletion or ROCK1 inhibition.…”

“…This hypothesis has been further supported by studies investigating the function of RNF20 and RNF40 (13–17). In contrast, we and others have uncovered tumor supportive roles of RNF20 and RNF40 in colorectal cancer (18,19) and androgen-dependent prostate cancer (20). Therefore, together these findings suggest that RNF20/RNF40-driven H2B monoubiquitination plays a context-dependent role in cancer.…”

Transcriptome-wide association study of multiple myeloma identifies candidate susceptibility genes

“…As such, genes identified within this TWAS build upon previously suggested candidate disease mechanisms which may confer MM predisposition [2], including anti-apoptotic effects, roles in DNA double-strand break repair and cell cycle regulation. Furthermore, many of the genes identified have been previously investigated in vitro for their roles in cancer and this adds further support as plausible candidate genes for MM predisposition [24,26,[30][31][32].…”

“…At 16p11.2, RNF40 is a promising candidate for MM susceptibility due to its role in double-strand break repair during homologous recombination (HR) and class switch recombination [28,29]. This gene has also been implicated in colorectal cancer [30]. A further candidate at this locus, QPRT has been demonstrated to confer resistance to chemotherapy and radiotherapy when studied in glioma and leukaemia [31,32].…”