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OTUB1 de-ubiquitinating enzyme promotes prostate cancer cell invasion in vitro and tumorigenesis in vivo.

Iglesias-Gato D, Chuan YC, Jiang N, Svensson C, Bao J, Paul I, Egevad L, Kessler BM, Wikström P, Niu Y, Flores-Morales A - Mol. Cancer (2015)

Bottom Line: Ubiquitination is a highly dynamic and reversible process with a central role in cell homeostasis.In localized prostate cancer tumors OTUB1 was found overexpressed as compared to normal prostatic epithelial cells.Our results suggest that drugs targeting the catalytic activity of OTUB1 could potentially be used as therapeutics for metastatic prostate cancer.

View Article: PubMed Central - PubMed

Affiliation: The Novo Nordisk Foundation Center for Protein Research, Faculty of Health Sciences, University of Copenhagen, 2200, Copenhagen, Denmark. diego.iglesias@cpr.ku.dk.

ABSTRACT

Background: Ubiquitination is a highly dynamic and reversible process with a central role in cell homeostasis. Deregulation of several deubiquitinating enzymes has been linked to tumor development but their specific role in prostate cancer progression remains unexplored.

Methods: RNAi screening was used to investigate the role of the ovarian tumor proteases (OTU) family of deubiquitinating enzymes on the proliferation and invasion capacity of prostate cancer cells. RhoA activity was measured in relation with OTUB1 effects on prostate cancer cell invasion. Tumor xenograft mouse model with stable OTUB1 knockdown was used to investigate OTUB1 influence in tumor growth.

Results: Our RNAi screening identified OTUB1 as an important regulator of prostate cancer cell invasion through the modulation of RhoA activation. The effect of OTUB1 on RhoA activation is important for androgen-induced repression of p53 expression in prostate cancer cells. In localized prostate cancer tumors OTUB1 was found overexpressed as compared to normal prostatic epithelial cells. Prostate cancer xenografts expressing reduced levels of OTUB1 exhibit reduced tumor growth and reduced metastatic dissemination in vivo.

Conclusions: OTUB1 mediates prostate cancer cell invasion through RhoA activation and promotes tumorigenesis in vivo. Our results suggest that drugs targeting the catalytic activity of OTUB1 could potentially be used as therapeutics for metastatic prostate cancer.

No MeSH data available.


Related in: MedlinePlus

Functions of the OTU-domain containing proteins in prostate cancer progression. (A) LNCaP-FGC cells transfected with siRNAs targeting different OTU family members were assayed for: (left panel) the knockdown efficiency of the siRNAs measured by quantitative real-time PCR -results are shown as percentage expression of each gene relative to siRNA control transfected cells; (middle panel) cell proliferation and (right panel) matrigel cell invasion. (B) Matrigel invasion assay (upper panel) and western blot analysis of OTUB1 expression (lower panel) in LNCaP-FGC cells transfected with two different siRNAs targeting OTUB1 or a control siRNA, treated with DHT (10 nM). (C) Same as in (B) using LNCaP-FGC cells transfected with either an empty vector or expression plasmids for wild type OTUB1 (OTUB1-WT) or the protease inactive C91S (OTUB1-C91S) variant. (D) Measurements of matrigel invasion and (E) cell proliferation of PC3 cells transfected with either siRNAs targeting OTUB1 or expression vectors bearing OTUB1-WT or the C91S variant, as indicated. In all panels, each column represents the average ± SD of at least four independent replicates and Student´s t test was used for statistical analysis. In (A), *indicates a significant reduction (p < 0.05) of mRNA levels and invasive capacity between LNCaP-FGC cells transfected with control siRNA compared with the different OTUs siRNAs. In (B) and (C), *indicates a statistically significant (p < 0.05) change after DHT treatment in cells transfected with the same siRNA or plasmid. #indicates a significant (p < 0.05) change between cells transfected with siOTUB1, OTUB1-WT or OTUB1-C91S and control transfected cells upon DHT stimulation. In (D), #indicates a significant (p < 0.05) change between cells transfected with siOTUB1, OTUB1-WT or OTUB1-C91S versus control transfected cells.
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Fig1: Functions of the OTU-domain containing proteins in prostate cancer progression. (A) LNCaP-FGC cells transfected with siRNAs targeting different OTU family members were assayed for: (left panel) the knockdown efficiency of the siRNAs measured by quantitative real-time PCR -results are shown as percentage expression of each gene relative to siRNA control transfected cells; (middle panel) cell proliferation and (right panel) matrigel cell invasion. (B) Matrigel invasion assay (upper panel) and western blot analysis of OTUB1 expression (lower panel) in LNCaP-FGC cells transfected with two different siRNAs targeting OTUB1 or a control siRNA, treated with DHT (10 nM). (C) Same as in (B) using LNCaP-FGC cells transfected with either an empty vector or expression plasmids for wild type OTUB1 (OTUB1-WT) or the protease inactive C91S (OTUB1-C91S) variant. (D) Measurements of matrigel invasion and (E) cell proliferation of PC3 cells transfected with either siRNAs targeting OTUB1 or expression vectors bearing OTUB1-WT or the C91S variant, as indicated. In all panels, each column represents the average ± SD of at least four independent replicates and Student´s t test was used for statistical analysis. In (A), *indicates a significant reduction (p < 0.05) of mRNA levels and invasive capacity between LNCaP-FGC cells transfected with control siRNA compared with the different OTUs siRNAs. In (B) and (C), *indicates a statistically significant (p < 0.05) change after DHT treatment in cells transfected with the same siRNA or plasmid. #indicates a significant (p < 0.05) change between cells transfected with siOTUB1, OTUB1-WT or OTUB1-C91S and control transfected cells upon DHT stimulation. In (D), #indicates a significant (p < 0.05) change between cells transfected with siOTUB1, OTUB1-WT or OTUB1-C91S versus control transfected cells.

Mentions: We wanted to investigate the potential roles of OTU-domain containing proteins with cysteine protease function (OTUD) in prostate cancer cells tumorigenesis. Therefore, we performed a small interfering RNA (siRNA)-based screening against a panel of OTU family members -OTUB1, OTUB2, OTUD3, OTUD4, OTUD5, OTUD7B and OTUD7C and TRABID- to measure their influence in the proliferation and invasion capacity of LNCaP-FGC cells. The efficiency of the knockdown was assessed by measuring the reduction of mRNA levels of each gene compared to scrambled siRNA transfected controls. After transfecting with the siRNA pools, at least 70% reduction was observed for all OTUD mRNAs but for OTUD7C mRNA (40%) (Figure 1A, left panel). Transient transfection of the aforementioned siRNAs into LNCaP-FGC cells didn’t result in a significant alteration of cell proliferation in vitro (Figure 1A, middle panel). LNCaP-FGC cells show a low capacity to invade through matrigel in vitro, which can be significantly stimulated by dihydrotestosterone (DHT) treatment [11]. Therefore, we tested the effects of the OTUD family targeting siRNAs in DHT-induced invasion capacity of LNCaP-FGC cells. We found that of all the siRNAs tested, only the inhibition of OTUB1 expression was able to significantly affect cell invasion of LNCaP-FGC cells in presence of DHT (Figure 1A, right panel).Figure 1


OTUB1 de-ubiquitinating enzyme promotes prostate cancer cell invasion in vitro and tumorigenesis in vivo.

Iglesias-Gato D, Chuan YC, Jiang N, Svensson C, Bao J, Paul I, Egevad L, Kessler BM, Wikström P, Niu Y, Flores-Morales A - Mol. Cancer (2015)

Functions of the OTU-domain containing proteins in prostate cancer progression. (A) LNCaP-FGC cells transfected with siRNAs targeting different OTU family members were assayed for: (left panel) the knockdown efficiency of the siRNAs measured by quantitative real-time PCR -results are shown as percentage expression of each gene relative to siRNA control transfected cells; (middle panel) cell proliferation and (right panel) matrigel cell invasion. (B) Matrigel invasion assay (upper panel) and western blot analysis of OTUB1 expression (lower panel) in LNCaP-FGC cells transfected with two different siRNAs targeting OTUB1 or a control siRNA, treated with DHT (10 nM). (C) Same as in (B) using LNCaP-FGC cells transfected with either an empty vector or expression plasmids for wild type OTUB1 (OTUB1-WT) or the protease inactive C91S (OTUB1-C91S) variant. (D) Measurements of matrigel invasion and (E) cell proliferation of PC3 cells transfected with either siRNAs targeting OTUB1 or expression vectors bearing OTUB1-WT or the C91S variant, as indicated. In all panels, each column represents the average ± SD of at least four independent replicates and Student´s t test was used for statistical analysis. In (A), *indicates a significant reduction (p < 0.05) of mRNA levels and invasive capacity between LNCaP-FGC cells transfected with control siRNA compared with the different OTUs siRNAs. In (B) and (C), *indicates a statistically significant (p < 0.05) change after DHT treatment in cells transfected with the same siRNA or plasmid. #indicates a significant (p < 0.05) change between cells transfected with siOTUB1, OTUB1-WT or OTUB1-C91S and control transfected cells upon DHT stimulation. In (D), #indicates a significant (p < 0.05) change between cells transfected with siOTUB1, OTUB1-WT or OTUB1-C91S versus control transfected cells.
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Fig1: Functions of the OTU-domain containing proteins in prostate cancer progression. (A) LNCaP-FGC cells transfected with siRNAs targeting different OTU family members were assayed for: (left panel) the knockdown efficiency of the siRNAs measured by quantitative real-time PCR -results are shown as percentage expression of each gene relative to siRNA control transfected cells; (middle panel) cell proliferation and (right panel) matrigel cell invasion. (B) Matrigel invasion assay (upper panel) and western blot analysis of OTUB1 expression (lower panel) in LNCaP-FGC cells transfected with two different siRNAs targeting OTUB1 or a control siRNA, treated with DHT (10 nM). (C) Same as in (B) using LNCaP-FGC cells transfected with either an empty vector or expression plasmids for wild type OTUB1 (OTUB1-WT) or the protease inactive C91S (OTUB1-C91S) variant. (D) Measurements of matrigel invasion and (E) cell proliferation of PC3 cells transfected with either siRNAs targeting OTUB1 or expression vectors bearing OTUB1-WT or the C91S variant, as indicated. In all panels, each column represents the average ± SD of at least four independent replicates and Student´s t test was used for statistical analysis. In (A), *indicates a significant reduction (p < 0.05) of mRNA levels and invasive capacity between LNCaP-FGC cells transfected with control siRNA compared with the different OTUs siRNAs. In (B) and (C), *indicates a statistically significant (p < 0.05) change after DHT treatment in cells transfected with the same siRNA or plasmid. #indicates a significant (p < 0.05) change between cells transfected with siOTUB1, OTUB1-WT or OTUB1-C91S and control transfected cells upon DHT stimulation. In (D), #indicates a significant (p < 0.05) change between cells transfected with siOTUB1, OTUB1-WT or OTUB1-C91S versus control transfected cells.
Mentions: We wanted to investigate the potential roles of OTU-domain containing proteins with cysteine protease function (OTUD) in prostate cancer cells tumorigenesis. Therefore, we performed a small interfering RNA (siRNA)-based screening against a panel of OTU family members -OTUB1, OTUB2, OTUD3, OTUD4, OTUD5, OTUD7B and OTUD7C and TRABID- to measure their influence in the proliferation and invasion capacity of LNCaP-FGC cells. The efficiency of the knockdown was assessed by measuring the reduction of mRNA levels of each gene compared to scrambled siRNA transfected controls. After transfecting with the siRNA pools, at least 70% reduction was observed for all OTUD mRNAs but for OTUD7C mRNA (40%) (Figure 1A, left panel). Transient transfection of the aforementioned siRNAs into LNCaP-FGC cells didn’t result in a significant alteration of cell proliferation in vitro (Figure 1A, middle panel). LNCaP-FGC cells show a low capacity to invade through matrigel in vitro, which can be significantly stimulated by dihydrotestosterone (DHT) treatment [11]. Therefore, we tested the effects of the OTUD family targeting siRNAs in DHT-induced invasion capacity of LNCaP-FGC cells. We found that of all the siRNAs tested, only the inhibition of OTUB1 expression was able to significantly affect cell invasion of LNCaP-FGC cells in presence of DHT (Figure 1A, right panel).Figure 1

Bottom Line: Ubiquitination is a highly dynamic and reversible process with a central role in cell homeostasis.In localized prostate cancer tumors OTUB1 was found overexpressed as compared to normal prostatic epithelial cells.Our results suggest that drugs targeting the catalytic activity of OTUB1 could potentially be used as therapeutics for metastatic prostate cancer.

View Article: PubMed Central - PubMed

Affiliation: The Novo Nordisk Foundation Center for Protein Research, Faculty of Health Sciences, University of Copenhagen, 2200, Copenhagen, Denmark. diego.iglesias@cpr.ku.dk.

ABSTRACT

Background: Ubiquitination is a highly dynamic and reversible process with a central role in cell homeostasis. Deregulation of several deubiquitinating enzymes has been linked to tumor development but their specific role in prostate cancer progression remains unexplored.

Methods: RNAi screening was used to investigate the role of the ovarian tumor proteases (OTU) family of deubiquitinating enzymes on the proliferation and invasion capacity of prostate cancer cells. RhoA activity was measured in relation with OTUB1 effects on prostate cancer cell invasion. Tumor xenograft mouse model with stable OTUB1 knockdown was used to investigate OTUB1 influence in tumor growth.

Results: Our RNAi screening identified OTUB1 as an important regulator of prostate cancer cell invasion through the modulation of RhoA activation. The effect of OTUB1 on RhoA activation is important for androgen-induced repression of p53 expression in prostate cancer cells. In localized prostate cancer tumors OTUB1 was found overexpressed as compared to normal prostatic epithelial cells. Prostate cancer xenografts expressing reduced levels of OTUB1 exhibit reduced tumor growth and reduced metastatic dissemination in vivo.

Conclusions: OTUB1 mediates prostate cancer cell invasion through RhoA activation and promotes tumorigenesis in vivo. Our results suggest that drugs targeting the catalytic activity of OTUB1 could potentially be used as therapeutics for metastatic prostate cancer.

No MeSH data available.


Related in: MedlinePlus