Abstract
BackgroundColorectal 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.MethodsWe 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.ResultsHuman 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.ConclusionsOur 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.
Highlights
Colorectal cancer (CRC) is the fourth leading cause of cancer-related deaths worldwide, and deciphering underlying molecular mechanism is essential
RING finger protein 40 (RNF40) knockdown reduces growth and tumorigenic features of CRC cell lines Our previous studies suggested that RNF40 might be required for the pro-proliferative behavior of colorectal cancer cells in vitro [15]
To test the general impact of RNF40 reduction on CRC cells, we examined the morphology of HCT116 and four additional CRC cell lines not previously tested following siRNA-mediated knockdown of RNF40 compared to control cells
Summary
Colorectal cancer (CRC) is the fourth leading cause of cancer-related deaths worldwide, and deciphering underlying molecular mechanism is essential. The formation of CRC is a multistep process initiated by a hyperproliferation of the intestinal epithelium, leading to the formation of pre-cancerous polyps and, adenomas and adenocarcinomas [2]. BIM, and HRK) and anti-apoptotic factors (e.g., BCL-2, BCL-xL, and MCL1) [5] These proteins control cytochrome-c release from the mitochondria, which results in the activation of a cascade of cysteine-aspartic proteases (caspases). As reviewed by Baig and colleagues [10], several therapeutic approaches and agents are currently being tested clinically, including BH3 mimetics, which target proteins of the BCL-2 family in order to trigger apoptosis
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