The E3 ubiquitin ligase RNF40 suppresses apoptosis in colorectal cancer cells

Deborah Schneider, Robert Lorenz Chua, Nicole Molitor, Feda H. Hamdan, Eva Maria Rettenmeier, Evangelos Prokakis, Vivek Kumar Mishra, Vijayalakshmi Kari, Florian Wegwitz, Steven Johnsen, Robyn Laura Kosinsky

Research output: Contribution to journalArticle

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.

Original languageEnglish (US)
Article number98
JournalClinical Epigenetics
Volume11
Issue number1
DOIs
StatePublished - Jul 2 2019

Fingerprint

Ubiquitin-Protein Ligases
Colorectal Neoplasms
Apoptosis
Proteins
Caspase 7
Caspase 3
Cell Cycle
Cell Line
Neoplasms
Messenger RNA
Propidium
Chromatin Immunoprecipitation
Annexin A5
Ligases
Caspases
Ubiquitin
Transcriptome
Histones
Cell Survival
Flow Cytometry

Keywords

  • Apoptosis
  • Caspases
  • Colorectal cancer
  • H2Bub1
  • RNF40

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics
  • Developmental Biology
  • Genetics(clinical)

Cite this

Schneider, D., Chua, R. L., Molitor, N., Hamdan, F. H., Rettenmeier, E. M., Prokakis, E., ... Kosinsky, R. L. (2019). The E3 ubiquitin ligase RNF40 suppresses apoptosis in colorectal cancer cells. Clinical Epigenetics, 11(1), [98]. https://doi.org/10.1186/s13148-019-0698-x

The E3 ubiquitin ligase RNF40 suppresses apoptosis in colorectal cancer cells. / Schneider, Deborah; Chua, Robert Lorenz; Molitor, Nicole; Hamdan, Feda H.; Rettenmeier, Eva Maria; Prokakis, Evangelos; Mishra, Vivek Kumar; Kari, Vijayalakshmi; Wegwitz, Florian; Johnsen, Steven; Kosinsky, Robyn Laura.

In: Clinical Epigenetics, Vol. 11, No. 1, 98, 02.07.2019.

Research output: Contribution to journalArticle

Schneider, D, Chua, RL, Molitor, N, Hamdan, FH, Rettenmeier, EM, Prokakis, E, Mishra, VK, Kari, V, Wegwitz, F, Johnsen, S & Kosinsky, RL 2019, 'The E3 ubiquitin ligase RNF40 suppresses apoptosis in colorectal cancer cells', Clinical Epigenetics, vol. 11, no. 1, 98. https://doi.org/10.1186/s13148-019-0698-x
Schneider D, Chua RL, Molitor N, Hamdan FH, Rettenmeier EM, Prokakis E et al. The E3 ubiquitin ligase RNF40 suppresses apoptosis in colorectal cancer cells. Clinical Epigenetics. 2019 Jul 2;11(1). 98. https://doi.org/10.1186/s13148-019-0698-x
Schneider, Deborah ; Chua, Robert Lorenz ; Molitor, Nicole ; Hamdan, Feda H. ; Rettenmeier, Eva Maria ; Prokakis, Evangelos ; Mishra, Vivek Kumar ; Kari, Vijayalakshmi ; Wegwitz, Florian ; Johnsen, Steven ; Kosinsky, Robyn Laura. / The E3 ubiquitin ligase RNF40 suppresses apoptosis in colorectal cancer cells. In: Clinical Epigenetics. 2019 ; Vol. 11, No. 1.
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AU - Chua, Robert Lorenz

AU - Molitor, Nicole

AU - Hamdan, Feda H.

AU - Rettenmeier, Eva Maria

AU - Prokakis, Evangelos

AU - Mishra, Vivek Kumar

AU - Kari, Vijayalakshmi

AU - Wegwitz, Florian

AU - Johnsen, Steven

AU - Kosinsky, Robyn Laura

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N2 - 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.

AB - 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.

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