Limits...
The deubiquitinase USP9X suppresses pancreatic ductal adenocarcinoma.

Pérez-Mancera PA, Rust AG, van der Weyden L, Kristiansen G, Li A, Sarver AL, Silverstein KA, Grützmann R, Aust D, Rümmele P, Knösel T, Herd C, Stemple DL, Kettleborough R, Brosnan JA, Li A, Morgan R, Knight S, Yu J, Stegeman S, Collier LS, ten Hoeve JJ, de Ridder J, Klein AP, Goggins M, Hruban RH, Chang DK, Biankin AV, Grimmond SM, Australian Pancreatic Cancer Genome InitiativeWessels LF, Wood SA, Iacobuzio-Donahue CA, Pilarsky C, Largaespada DA, Adams DJ, Tuveson DA - Nature (2012)

Bottom Line: Although previous work had attributed a pro-survival role to USP9X in human neoplasia, we found instead that loss of Usp9x enhances transformation and protects pancreatic cancer cells from anoikis.Clinically, low USP9X protein and messenger RNA expression in PDA correlates with poor survival after surgery, and USP9X levels are inversely associated with metastatic burden in advanced disease.Furthermore, chromatin modulation with trichostatin A or 5-aza-2'-deoxycytidine elevates USP9X expression in human PDA cell lines, indicating a clinical approach for certain patients.

View Article: PubMed Central - PubMed

Affiliation: Li Ka Shing Centre, Cambridge Research Institute, Cancer Research UK, Cambridge CB2 0RE, UK.

ABSTRACT
Pancreatic ductal adenocarcinoma (PDA) remains a lethal malignancy despite much progress concerning its molecular characterization. PDA tumours harbour four signature somatic mutations in addition to numerous lower frequency genetic events of uncertain significance. Here we use Sleeping Beauty (SB) transposon-mediated insertional mutagenesis in a mouse model of pancreatic ductal preneoplasia to identify genes that cooperate with oncogenic Kras(G12D) to accelerate tumorigenesis and promote progression. Our screen revealed new candidate genes for PDA and confirmed the importance of many genes and pathways previously implicated in human PDA. The most commonly mutated gene was the X-linked deubiquitinase Usp9x, which was inactivated in over 50% of the tumours. Although previous work had attributed a pro-survival role to USP9X in human neoplasia, we found instead that loss of Usp9x enhances transformation and protects pancreatic cancer cells from anoikis. Clinically, low USP9X protein and messenger RNA expression in PDA correlates with poor survival after surgery, and USP9X levels are inversely associated with metastatic burden in advanced disease. Furthermore, chromatin modulation with trichostatin A or 5-aza-2'-deoxycytidine elevates USP9X expression in human PDA cell lines, indicating a clinical approach for certain patients. The conditional deletion of Usp9x cooperated with Kras(G12D) to accelerate pancreatic tumorigenesis in mice, validating their genetic interaction. We propose that USP9X is a major tumour suppressor gene with prognostic and therapeutic relevance in PDA.

Show MeSH

Related in: MedlinePlus

Usp9x regulates PDA cellular transformation and Itcha–b, Usp9x knock-down promotes anchorage-independent growth in three mPDA cell lines (a), and decreases anoikis denoted by cleaved caspase 3 (CC3) (b). The mean and s.e.m. of one representative experiment performed in triplicate are shown in (a) (***, p<0.001; Mann Whitney test). (S: Scramble; U: Usp9x). c, Usp9x knock-down decreases Itch but not Ask1 or Mcl1. Changes in Itch are more evident in suspension cultures, and the slower migrating band has the expected mobility of mono-ubiquitinated Itch. d, Ectopic Itch induces anoikis. (B: pBabe-neo; I: pBabe-neo-myc-Itch).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3376394&req=5

Figure 2: Usp9x regulates PDA cellular transformation and Itcha–b, Usp9x knock-down promotes anchorage-independent growth in three mPDA cell lines (a), and decreases anoikis denoted by cleaved caspase 3 (CC3) (b). The mean and s.e.m. of one representative experiment performed in triplicate are shown in (a) (***, p<0.001; Mann Whitney test). (S: Scramble; U: Usp9x). c, Usp9x knock-down decreases Itch but not Ask1 or Mcl1. Changes in Itch are more evident in suspension cultures, and the slower migrating band has the expected mobility of mono-ubiquitinated Itch. d, Ectopic Itch induces anoikis. (B: pBabe-neo; I: pBabe-neo-myc-Itch).

Mentions: To characterize the cellular and molecular pathways affected by Usp9x in PDA, RNAi was used to deplete Usp9x in mPDA cell lines (Supplementary Fig. 5a). While Usp9x depletion did not affect the proliferation of monolayer cultures (Supplementary Fig. 5b), it significantly increased colony formation in soft agar (Fig. 2a, Supplementary Fig. 5c), compared to cells transfected with scrambled shRNAs. Additionally, Usp9x knock-down potently suppressed anoikis in mPDA cells (Fig. 2b). These properties of Usp9x were predominantly dependent upon its intrinsic deubiquitinase activity (Supplementary Fig. 6a, b).


The deubiquitinase USP9X suppresses pancreatic ductal adenocarcinoma.

Pérez-Mancera PA, Rust AG, van der Weyden L, Kristiansen G, Li A, Sarver AL, Silverstein KA, Grützmann R, Aust D, Rümmele P, Knösel T, Herd C, Stemple DL, Kettleborough R, Brosnan JA, Li A, Morgan R, Knight S, Yu J, Stegeman S, Collier LS, ten Hoeve JJ, de Ridder J, Klein AP, Goggins M, Hruban RH, Chang DK, Biankin AV, Grimmond SM, Australian Pancreatic Cancer Genome InitiativeWessels LF, Wood SA, Iacobuzio-Donahue CA, Pilarsky C, Largaespada DA, Adams DJ, Tuveson DA - Nature (2012)

Usp9x regulates PDA cellular transformation and Itcha–b, Usp9x knock-down promotes anchorage-independent growth in three mPDA cell lines (a), and decreases anoikis denoted by cleaved caspase 3 (CC3) (b). The mean and s.e.m. of one representative experiment performed in triplicate are shown in (a) (***, p<0.001; Mann Whitney test). (S: Scramble; U: Usp9x). c, Usp9x knock-down decreases Itch but not Ask1 or Mcl1. Changes in Itch are more evident in suspension cultures, and the slower migrating band has the expected mobility of mono-ubiquitinated Itch. d, Ectopic Itch induces anoikis. (B: pBabe-neo; I: pBabe-neo-myc-Itch).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3376394&req=5

Figure 2: Usp9x regulates PDA cellular transformation and Itcha–b, Usp9x knock-down promotes anchorage-independent growth in three mPDA cell lines (a), and decreases anoikis denoted by cleaved caspase 3 (CC3) (b). The mean and s.e.m. of one representative experiment performed in triplicate are shown in (a) (***, p<0.001; Mann Whitney test). (S: Scramble; U: Usp9x). c, Usp9x knock-down decreases Itch but not Ask1 or Mcl1. Changes in Itch are more evident in suspension cultures, and the slower migrating band has the expected mobility of mono-ubiquitinated Itch. d, Ectopic Itch induces anoikis. (B: pBabe-neo; I: pBabe-neo-myc-Itch).
Mentions: To characterize the cellular and molecular pathways affected by Usp9x in PDA, RNAi was used to deplete Usp9x in mPDA cell lines (Supplementary Fig. 5a). While Usp9x depletion did not affect the proliferation of monolayer cultures (Supplementary Fig. 5b), it significantly increased colony formation in soft agar (Fig. 2a, Supplementary Fig. 5c), compared to cells transfected with scrambled shRNAs. Additionally, Usp9x knock-down potently suppressed anoikis in mPDA cells (Fig. 2b). These properties of Usp9x were predominantly dependent upon its intrinsic deubiquitinase activity (Supplementary Fig. 6a, b).

Bottom Line: Although previous work had attributed a pro-survival role to USP9X in human neoplasia, we found instead that loss of Usp9x enhances transformation and protects pancreatic cancer cells from anoikis.Clinically, low USP9X protein and messenger RNA expression in PDA correlates with poor survival after surgery, and USP9X levels are inversely associated with metastatic burden in advanced disease.Furthermore, chromatin modulation with trichostatin A or 5-aza-2'-deoxycytidine elevates USP9X expression in human PDA cell lines, indicating a clinical approach for certain patients.

View Article: PubMed Central - PubMed

Affiliation: Li Ka Shing Centre, Cambridge Research Institute, Cancer Research UK, Cambridge CB2 0RE, UK.

ABSTRACT
Pancreatic ductal adenocarcinoma (PDA) remains a lethal malignancy despite much progress concerning its molecular characterization. PDA tumours harbour four signature somatic mutations in addition to numerous lower frequency genetic events of uncertain significance. Here we use Sleeping Beauty (SB) transposon-mediated insertional mutagenesis in a mouse model of pancreatic ductal preneoplasia to identify genes that cooperate with oncogenic Kras(G12D) to accelerate tumorigenesis and promote progression. Our screen revealed new candidate genes for PDA and confirmed the importance of many genes and pathways previously implicated in human PDA. The most commonly mutated gene was the X-linked deubiquitinase Usp9x, which was inactivated in over 50% of the tumours. Although previous work had attributed a pro-survival role to USP9X in human neoplasia, we found instead that loss of Usp9x enhances transformation and protects pancreatic cancer cells from anoikis. Clinically, low USP9X protein and messenger RNA expression in PDA correlates with poor survival after surgery, and USP9X levels are inversely associated with metastatic burden in advanced disease. Furthermore, chromatin modulation with trichostatin A or 5-aza-2'-deoxycytidine elevates USP9X expression in human PDA cell lines, indicating a clinical approach for certain patients. The conditional deletion of Usp9x cooperated with Kras(G12D) to accelerate pancreatic tumorigenesis in mice, validating their genetic interaction. We propose that USP9X is a major tumour suppressor gene with prognostic and therapeutic relevance in PDA.

Show MeSH
Related in: MedlinePlus