The deubiquitinase USP9X suppresses pancreatic ductal adenocarcinoma

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.

Figure 1. Transposon mutagenesis accelerates murine PDA and targets Usp9x

a, 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.

Figure 2. Usp9x regulates PDA cellular transformation and Itch

a–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).

Figure 3. USP9X loss promotes PDA

a–c, Decreased USP9X expression correlates with shortened survival in Australian post-surgical cohort (a) (8.7 vs. 18.4 months, p=0.0076; log-rank test), increased metastatic burden in American autopsy series (b) (54% vs. 19%, p=0.0212; Fisher’s exact test), and diminished survival in German post-surgical cohort (c) (11.1 vs. 16.1 months, p=0.037; log-rank test). d, Trichostatin A (TSA, Red) and 5-Aza-2′-deoxycytidine (AZA, Blue) modestly increase USP9X mRNA expression. The mean and s.e.m. of one representative experiment performed in triplicate are shown. e, Usp9x deletion promotes mPanIN progression in KCU mice (p<0.0001; Fisher’s exact test). f, Representative normal pancreas (CU), mPanIN1 (KC), mPanIN3 (KCU) and microinvasive mPDA (KCU, arrow, circled). Scale bar: 100μm.