Usp9x Promotes Survival in Human Pancreatic Cancer and Its Inhibition Suppresses Pancreatic Ductal Adenocarcinoma In Vivo Tumor Growth - PubMed (original) (raw)

Usp9x Promotes Survival in Human Pancreatic Cancer and Its Inhibition Suppresses Pancreatic Ductal Adenocarcinoma In Vivo Tumor Growth

Anupama Pal et al. Neoplasia. 2018 Feb.

Abstract

Usp9x has emerged as a potential therapeutic target in some hematologic malignancies and a broad range of solid tumors including brain, breast, and prostate. To examine Usp9x tumorigenicity and consequence of Usp9x inhibition in human pancreatic tumor models, we carried out gain- and loss-of-function studies using established human pancreatic tumor cell lines (PANC1 and MIAPACA2) and four spontaneously immortalized human pancreatic patient-derived tumor (PDX) cell lines. The effect of Usp9x activity inhibition by small molecule deubiquitinase inhibitor G9 was assessed in 2D and 3D culture, and its efficacy was tested in human tumor xenografts. Overexpression of Usp9x increased 3D growth and invasion in PANC1 cells and up-regulated the expression of known Usp9x substrates Mcl-1 and ITCH. Usp9x inhibition by shRNA-knockdown or by G9 treatment reduced 3D colony formation in PANC1 and PDX cell lines, induced rapid apoptosis in MIAPACA2 cells, and associated with reduced Mcl-1 and ITCH protein levels. Although G9 treatment reduced human MIAPACA2 tumor burden in vivo, in mouse pancreatic cancer cell lines established from constitutive (8041) and doxycycline-inducible (4668) KrasG12D/Tp53R172H mouse pancreatic tumors, Usp9x inhibition increased and sustained the 3D colony growth and showed no significant effect on tumor growth in 8041-xenografts. Thus, Usp9x inhibition may be therapeutically active in human PDAC, but this activity was not predicted from studies of genetically engineered mouse pancreatic tumor models.

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Figures

Figure 1

Overexpression of Usp9x increases 3D colony formation and invasion of human PANC1 cells. (A). Immunoblot analysis confirms expression of Flag-tagged Usp9x in human PDAC cell line PANC1. Usp9x-OV increases protein expression levels of Usp9x substrates ITCH and Mcl-1. β-Actin was used as the loading control. (B) Phase contrast images show effect of Usp9x overexpression on the growth of PANC1 cells in monolayer. PANC1-Usp9x-OV cells show a fibroblast-like phenotype in monolayer cultures as compared to epithelial phenotype of PANC1-vector control cells. (C) Phase contrast images show effect of Usp9x overexpression on the growth of PANC1 cells in 3D culture on 2% GFR Matrigel. PANC1-Usp9x-OV cells show a typical malignant phenotype with disruption of flat, round 3D colony growth as compared to PANC1-vector control cells. Representative column graph shows an increase in the number of colonies formed in 3D culture in PANC1-Usp9x-OV cells compared to PANC1-vector control cells. **P < .005. (D) Phase contrast images show increased spheroid size in suspension culture in PANC1-Usp9x-OV as compared with PANC1-vector control cells. Representative column graph shows an increase in the size of spheroids grown in suspension culture in PANC1-Usp9x-OV cells compared to PANC1-vector control cells. ****P < .00005. (E) Representative column graph shows increased invasion in PANC1-Usp9x-OV cells through GFR Matrigel invasion chambers as compared to PANC1-vector control cells. *P < .05.

Figure 2

Inhibition of Usp9x inhibits 3D colony formation in established human PDAC cell line PANC1 and newly established human pancreatic patient-derived cell lines. (A) Representative column graph shows a 50% decrease in number of colonies formed in 3D culture in PANC1-Usp9x-KD cells as compared to PANC1-vector control cells. *P < .05. Immunoblot analysis confirms Usp9x KD in PANC1 cells. (B) Immunoblot analysis confirms Usp9x KD in 4 human pancreatic cancer cell lines derived from pancreatic tumor patient samples: UM-2, UM-6, UM-16, and UM-76. (C) Phase contrast images of control and Usp9x-KD UM-2, UM-6, UM-16, and UM-76 cells grown on 2% GFR Matrigel in 3D culture for 8 days. UM-2-Usp9x KD, UM-6-Usp9x KD, UM-16-Usp9x KD, and UM-76-Usp9x KD show complete inhibition of colony formation in 3D culture as compared to respective scrambled control cells. (D) MTT assay shows inhibition of cell growth and survival in UM-2, UM-6, UM-16, and UM-76 cells following treatment with small-molecule deubiquitinase inhibitor G9. Cells were grown in triplicates in 96-well culture clusters for 24 hours followed by treatment with serially diluted dose levels of G9. Cell growth/survival was determined after 48 hours of treatment. The average ± S.D. of triplicate assays is shown. (E) Phase contrast images show inhibition of colony formation in G9-treated UM-2 cells. UM-2 cells were treated with vehicle or 600 nM G9 every 3 days while growing in 3D conditions for 8 days.

Figure 3

Usp9x KD promotes growth, while Usp9x overexpression reduces growth, in 3D culture of mutant KrasG12D pancreatic mouse xenograft cell lines with constitutive expression of KrasG12D and Tp53R172H. (A) Immunoblot analysis confirms Usp9x KD in three mutant KrasG12D pancreatic mouse xenograft cell lines with constitutive expression of KrasG12D and Tp53R172H: 8041, 65671, and 13442. Representative column graphs show that 8041-Usp9x-KD1 and 8041-Usp9x-KD2, 65671-Usp9x-KD1 and 65671-Usp9x-KD2, and 13442-Usp9x-KD1 cells form increased numbers of colonies as compared to their respective scrambled control cells when grown on 2% Matrigel for 8 days in 3D culture. *P < .05, **P < .005. (B) Representative column graph shows that treatment of 8041 cells with small-molecule inhibitor G9 at 600 nM lea to formation of an increased number of colonies as compared to vehicle-treated controls when grown on 2% Matrigel for 8 days in 3D culture. *P < .05. (C) Phase contrast images show that 8041-Usp9x-KD1 cells form larger spheroidal structures as compared to scrambled control cells when grown in suspension culture on agarose plates. A total of 106 cells of 8041-control or 8041-Usp9x- KD1 cells were plated on agarose plates and allowed to grow in suspension for 5 days. Representative column graph shows an increase in the size of spheroids grown in suspension culture in 8041-Usp9x- KD1 cells as compared to 8041-control. **P < .005 (D) Immunoblot analysis confirms expression of Flag-tagged Usp9x in murine 8041 cells. The expression levels of ITCH and Mcl-1 remain unaltered in 8041-Usp9x-OV cells as compared to vector controls. β-Actin was used as the loading control. (E) Representative column graph shows that 8041-Usp9x-OV cells form a decreased number of colonies as compared to 8041-vector control cells when grown on 2% Matrigel for 8 days in 3D culture. (F) Phase contrast images show the effect of Usp9x overexpression on the growth of 8041 cells as spheroids in suspension culture. Usp9x expression in 8041 cells shows decreased growth as compared to vector controls. Representative column graph shows a decrease in the size of spheroids grown in suspension culture in 8041-Usp9x-OV cells as compared to 8041-Vector Control cells. **P < .005.

Figure 4

Usp9x promotes growth in 3D culture independent of mutant Kras in the mutant KrasG12D pancreatic mouse xenograft cell line with inducible expression of KrasG12D and Tp53R172H. (A) Immunoblot analysis shows no change in the activity and total protein levels of Usp9x in DOX depleted 4668 cells with withdrawn (−) KrasG12D support as compared to controls with sustained (+) DOX support. 4668 cells with DOX-inducible KrasG12D expression were DOX depleted for 48 hours followed by DUB activity assessment by DUB activity assay using HA-UbVS. The samples were subjected to immunoblot analysis with anti-HA antibody to detect HA-UbVS–labeled Usp9x (Activity). Inhibited phospho-ERK1/2 levels confirm withdrawal of KrasG12D support in DOX-depleted 4668 cells. β-Actin was used as the loading control. (B) RT-PCR analysis of 4668 cells show no change in the transcript levels of Usp9x in the absence (−) or presence (+) of DOX (KrasG12D expression) for 48 hours. Average ± S.D. of triplicate assays is shown. (C) Phase contrast images show that the inhibition of mutant KrasG12D activity mediated by DOX depletion in inducible 4668 mouse pancreatic tumor cells inhibits colony formation on 2% GFR Matrigel in 3D culture. The 3D colony formation was rescued by retreatment with DOX. No effect of DOX depletion was observed on cell growth in 2D monolayers. 4668 cells were grown with or without DOX as 2D monolayer (top) for 48 hours before plating on 2% Matrigel with continued or depleted DOX condition in 3D (bottom). At day 4, DOX depletion was rescued by adding DOX back for another 4 days. Phase contrast images were acquired at day 8 of 3D culture. (D) Immunoblot analysis shows that Usp9x activity and protein levels are only nominally affected in 4668 cells in the sustained presence or absence of KrasG12D support by DOX addition or depletion. (E) Immunoblot analysis confirms Usp9x KD in 4668-Usp9x-KD1 and 4668-Usp9x-KD2 cells as compared to scrambled 4668-control cells. (F) Phase Contrast images show that DOX-depleted and Kras activity-inhibited 4668-Usp9x-KD1 and 4668-Usp9x-KD2 cells form aggressive colonies in 3D culture as compared to DOX-depleted 4668-control cells that show disintegration of colony formation.

Figure 5

Usp9x inhibition induces apoptosis in Kras mutant human cell line MIAPACA2, but Kras w/t BxPC3 cells remain unaffected. (A) Immunoblot analysis shows that Usp9x KD in human mutant Kras PDAC cell line MIAPACA2 reduces mutant Kras expression levels, whereas Usp9x KD in the mouse mutant Kras cell line 8041 has no effect on Kras. (B) Immunoblot analysis confirms Usp9x KD in human PDAC cell lines MIAPACA2 and BxPC3 with mutant Kras and w/t Kras, respectively. Usp9x KD in MIAPACA2 cells led to rapid apoptosis induction as indicated by PARP cleavage, but no such effect was observed in the w/t Kras cell line BxPC3. (C) Phase contrast images further confirm apoptosis induction in MIAPACA2-Usp9x KD cells as compared to MIAPACA2-Control cells. No effect of Usp9x KD on the phenotype of BxPC3 cells grown in monolayer was observed. (D) Representative column graph shows that BxPC3-Usp9x-KD cells show no effect on colony formation on 2% Matrigel in 3D culture when compared to BxPC3-Control cells. n.s ., not significant.

Figure 6

Small-molecule inhibitor G9 decreases tumor size in human MIAPACA2 xenografts but has no effect on murine 8041 xenografts. (A) G9 treatment significantly reduces tumor size in human MIAPACA2 xenografts. N = 4 for each group. *P < .05, **P < .005. A total of 5 × 106 MIAPACA2 cells were injected subcutaneously, mid-dorsally in NSG mice. When measurable, tumors were size matched and treated i.p. with either vehicle or 15 mg/kg of G9 every other day for 12 days. Tumor size was monitored, and tumor measurements were recorded. (B) Immunoblot analysis shows inhibited activity of Usp9x in G9-treated human MIAPACA2-xenograft tumors. Tumor tissue was extracted from vehicle- or G9-treated mice and lysed in DUB assay buffer before incubation with HA-UbVS and immunoblot analysis with anti-HA antibody to detect HA-UbVS–labeled DUBs as a measure of DUB activity (Usp9x activity is denoted). β-Actin was used as the loading control. (C) G9 treatment showed no significant effect on growth of murine 8041 xenograft tumors. Tumors were established with 2 × 106 8041 cells as described in A. N = 5 for each group. (D) Immunoblot analysis shows inhibition of Usp9x activity in G9-treated murine 8041-xenograft tumors. β-Actin was used as the loading control.

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Usp9x Promotes Survival in Human Pancreatic Cancer and Its Inhibition Suppresses Pancreatic Ductal Adenocarcinoma In Vivo Tumor Growth - PubMed (original) (raw)