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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.

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Transposon mutagenesis accelerates murine PDA and targets Usp9xa, Increased mortality of KCTSB13 mice compared to KC cohort (containing KCT, KCSB13 and KC mice) (172 vs. 257 days, p<0.001; long-rank test). Wild-type (WT) cohort is comprised of KTSB13 and CTSB13 mice. b–c, Invasive cystic neoplasm (b), and mPDA (c) in KCTSB13 mice. Scale bar: 100μm. d, Usp9x is the major CIS in KCTSB13 PDA tumors (X-axis denotes genome, Y-axis −log P-value), with bidirectional insertions. (+) parallel to Usp9x expression, (−) antiparallel. e, Usp9x Exon 2-T2/Onc chimeric mRNA in SB13 tumors. f–g, Usp9x protein expression in normal pancreatic ducts (arrow) (f), but not in neoplastic cells (g) (arrows) in SB13 PDA harboring Usp9x insertions. Scale bar: 100μm.
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Figure 1: Transposon mutagenesis accelerates murine PDA and targets Usp9xa, Increased mortality of KCTSB13 mice compared to KC cohort (containing KCT, KCSB13 and KC mice) (172 vs. 257 days, p<0.001; long-rank test). Wild-type (WT) cohort is comprised of KTSB13 and CTSB13 mice. b–c, Invasive cystic neoplasm (b), and mPDA (c) in KCTSB13 mice. Scale bar: 100μm. d, Usp9x is the major CIS in KCTSB13 PDA tumors (X-axis denotes genome, Y-axis −log P-value), with bidirectional insertions. (+) parallel to Usp9x expression, (−) antiparallel. e, Usp9x Exon 2-T2/Onc chimeric mRNA in SB13 tumors. f–g, Usp9x protein expression in normal pancreatic ducts (arrow) (f), but not in neoplastic cells (g) (arrows) in SB13 PDA harboring Usp9x insertions. Scale bar: 100μm.

Mentions: To increase the specificity and potency of SB mutagenesis, we generated a conditional SB13 mutant mouse by targeting the Rosa26 locus in embryonic stem cells (Supplementary Fig. 3a, b). The pancreatic specific expression and function of the conditional SB13 allele was confirmed (Supplementary Fig. 3c), and we found that SB13-induced transposition by itself did not promote lethality or pancreatic tumorigenesis (Fig. 1a, Supplementary Fig. 4a). In contrast, KCTSB13 mice (KrasLSL-G12D; Pdx1-cre; T2/Onc; Rosa26-LSL-SB13) rapidly progressed and succumbed to invasive pancreatic neoplasms (Fig. 1a–c). A cohort of 117 KCTSB13 mice (Supplementary Fig. 1b) was monitored for tumor development, and 103 of these mice were available for full necropsy and tissue procurement. The majority of such mice harbored multi-focal pancreatic tumors, and 198 distinct primary tumors and metastases were subjected to histological and molecular analysis. Most mice had invasive carcinomas (66/103) that consisted of classical mPDA (78.8%) or invasive cystic neoplasms (21.2%); 34.8% of mice also contained metastases predominantly in their liver and lungs (Supplementary Fig. 4c). The remainder of the mice (37/103) had preinvasive pancreatic tumors consisting of high grade mPanIN and cyst-forming papillary neoplasms (Supplementary Fig. 4b).


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)

Transposon mutagenesis accelerates murine PDA and targets Usp9xa, Increased mortality of KCTSB13 mice compared to KC cohort (containing KCT, KCSB13 and KC mice) (172 vs. 257 days, p<0.001; long-rank test). Wild-type (WT) cohort is comprised of KTSB13 and CTSB13 mice. b–c, Invasive cystic neoplasm (b), and mPDA (c) in KCTSB13 mice. Scale bar: 100μm. d, Usp9x is the major CIS in KCTSB13 PDA tumors (X-axis denotes genome, Y-axis −log P-value), with bidirectional insertions. (+) parallel to Usp9x expression, (−) antiparallel. e, Usp9x Exon 2-T2/Onc chimeric mRNA in SB13 tumors. f–g, Usp9x protein expression in normal pancreatic ducts (arrow) (f), but not in neoplastic cells (g) (arrows) in SB13 PDA harboring Usp9x insertions. Scale bar: 100μm.
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Related In: Results  -  Collection

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Figure 1: Transposon mutagenesis accelerates murine PDA and targets Usp9xa, Increased mortality of KCTSB13 mice compared to KC cohort (containing KCT, KCSB13 and KC mice) (172 vs. 257 days, p<0.001; long-rank test). Wild-type (WT) cohort is comprised of KTSB13 and CTSB13 mice. b–c, Invasive cystic neoplasm (b), and mPDA (c) in KCTSB13 mice. Scale bar: 100μm. d, Usp9x is the major CIS in KCTSB13 PDA tumors (X-axis denotes genome, Y-axis −log P-value), with bidirectional insertions. (+) parallel to Usp9x expression, (−) antiparallel. e, Usp9x Exon 2-T2/Onc chimeric mRNA in SB13 tumors. f–g, Usp9x protein expression in normal pancreatic ducts (arrow) (f), but not in neoplastic cells (g) (arrows) in SB13 PDA harboring Usp9x insertions. Scale bar: 100μm.
Mentions: To increase the specificity and potency of SB mutagenesis, we generated a conditional SB13 mutant mouse by targeting the Rosa26 locus in embryonic stem cells (Supplementary Fig. 3a, b). The pancreatic specific expression and function of the conditional SB13 allele was confirmed (Supplementary Fig. 3c), and we found that SB13-induced transposition by itself did not promote lethality or pancreatic tumorigenesis (Fig. 1a, Supplementary Fig. 4a). In contrast, KCTSB13 mice (KrasLSL-G12D; Pdx1-cre; T2/Onc; Rosa26-LSL-SB13) rapidly progressed and succumbed to invasive pancreatic neoplasms (Fig. 1a–c). A cohort of 117 KCTSB13 mice (Supplementary Fig. 1b) was monitored for tumor development, and 103 of these mice were available for full necropsy and tissue procurement. The majority of such mice harbored multi-focal pancreatic tumors, and 198 distinct primary tumors and metastases were subjected to histological and molecular analysis. Most mice had invasive carcinomas (66/103) that consisted of classical mPDA (78.8%) or invasive cystic neoplasms (21.2%); 34.8% of mice also contained metastases predominantly in their liver and lungs (Supplementary Fig. 4c). The remainder of the mice (37/103) had preinvasive pancreatic tumors consisting of high grade mPanIN and cyst-forming papillary neoplasms (Supplementary Fig. 4b).

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