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. 2017 Dec 11;32(6):824-839.e8.
doi: 10.1016/j.ccell.2017.10.011. Epub 2017 Nov 16.

Stress-Activated NRF2-MDM2 Cascade Controls Neoplastic Progression in Pancreas

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Free PMC article

Stress-Activated NRF2-MDM2 Cascade Controls Neoplastic Progression in Pancreas

Jelena Todoric et al. Cancer Cell. .
Free PMC article

Abstract

Despite expression of oncogenic KRAS, premalignant pancreatic intraepithelial neoplasia 1 (PanIN1) lesions rarely become fully malignant pancreatic ductal adenocarcinoma (PDAC). The molecular mechanisms through which established risk factors, such as chronic pancreatitis, acinar cell damage, and/or defective autophagy increase the likelihood of PDAC development are poorly understood. We show that accumulation of the autophagy substrate p62/SQSTM1 in stressed KrasG12D acinar cells is associated with PDAC development and maintenance of malignancy in human cells and mice. p62 accumulation promotes neoplastic progression by controlling the NRF2-mediated induction of MDM2, which acts through p53-dependent and -independent mechanisms to abrogate checkpoints that prevent conversion of differentiated acinar cells to proliferative ductal progenitors. MDM2 targeting may be useful for preventing PDAC development in high-risk individuals.

Keywords: IKKα; MDM2; NRF2; acinar cell reprogramming; impaired autophagy; p62; pancreatic ductal adenocarcinoma.

Figures

Figure 1
Figure 1. p62 Accumulates in Human PDAC and Maintains Malignancy
(A) Representative immunohistochemical (IHC) analysis of normal human pancreata, pancreatitis and malignant tissues containing PanIN2/3 and PDAC with antibodies to indicated proteins. Scale bars: 25 µM. (B) Expression of indicated proteins in MIA PaCa-2 xenografts was examined by immunoblotting (IB) (n = 6). (C) Growth of MIA PaCa-2 xenografts transduced with either p62 or scrambled shRNA (n = 6). (D) Sphere formation by control and p62- ablated MIA PaCa-2 and Capan-2 cells (n = 6). (E) Sphere formation by control, IKKα-depleted and p62-ablated/IKKα-depleted MIA PaCa-2 and Capan-2 cells (n = 6). (F) Tumorigenic growth of control and IKKα-depleted MIA PaCa-2 cells. (G) Sphere formation by control and NRF2-ablated MIA PaCa-2 and Capan-2 cells (n = 6). (H) Tumorigenic growth of control and NRF2-ablated MIA PaCa-2 cells. p62, IKKα, and NRF2 ablation was examined by IB. Results in C–H are mean ± SEM; *, p < 0.05; **, p < 0.01; ***, p < 0.001. Statistical significance was calculated using Student’s t test (C–H). See also Figure S1.
Figure 2
Figure 2. Chronic Pancreatitis Enhances PanIN to PDAC Progression and p62 Accumulation
(A) Gross morphology of pancreata from KrasG12D and KrasG12D;IkkαΔpan mice, whose age is indicated. Arrow – pancreas. (B) H&E and amylase stained pancreatic sections from above mice. Scale bars: 100 µm. (C) Tumor latency (n = 6). (D) Kaplan-Meier survival curves (n = 6). (E) IB analysis of pancreatic lysates from 5-week-old mice. (F) Q-RT-PCR of Sqstm1 mRNA in 5-week-old mice of indicated genotypes (n = 5). (G) Q-RT-PCR of NRF2 target genes in RNA isolated from acinar and ductal cells of 5-week-old mice of indicated genotypes (n = 5). (H, I) Quantification of lesions per high power (200×) field (HPF) in 5- and 8-week-old mice of indicated genotypes (n = 5) for ADM (H) and PanIN (I). (J) Quantification of Ki67 staining in pancreata of 5-week-old mice. (K) IB analysis of pancreatic lysates. Loading control (ERK) is in panel E. (L) IHC of Sox9 and CK19 in pancreata of 5-week-old mice. Scale bars: 25 µm. (M) IHC of NQO1, MDM2 and HES1 in ductal progenitor lesions of 5-week-old mice. Scale bars: 25 µm. (N) p53 IB of pancreatic lysates from 5-week-old mice. Results in C and F–J are mean ± SEM; **, p < 0.01; ***, p < 0.001; ND, not detected. Statistical significance was calculated using Student’s t test (C, F–J) or log-rank test (D). See also Figure S2.
Figure 3
Figure 3. p62 Ablation Attenuates Pancreatitis-Accelerated Neoplastic Progression
(A) Quantification of ADM and PanIN1 density, amylase and Ki67 staining of pancreatic sections of indicated 5-week-old mice (n = 5). (B) H&E staining, amylase IHC, amylase and CK19 co-IF, and Sox9 IHC of pancreatic sections from indicated 5-week-old mice. Scale bars: 25 µm. (C) Kaplan-Meier survival curves of indicated mouse strains (n = 10). (D) Q-RT-PCR analysis of mRNA in acinar and ductal cell fractions from indicated 5-week-old mice (n = 5). (E) NQO1, MDM2 and HES1 IHC of pancreatic sections from indicated 5-week-old mice. Scale bars: 25 µm. (F) p53 IB analysis of pancreatic lysates from 5-week-old mice of indicated genotypes. (G) Frequency of ALDH expression in EpCAM+ cells from 8-week-old KrasG12D (n = 3), KrasG12D;IkkαΔpan (n = 7), and KrasG12D;Ikkα/p62Δpan (n = 4) mice. (H) Sphere-forming capacity of isolated ALDH+ cells from indicated genotypes (n = 3 per group). (I) Sphere formation of control and p62-ablated MIA PaCa-2 and Capan-2 cells with or without NICD1 overexpression. (J) Representative images and quantified SA-β-gal staining and γ-H2AX and ERK1/2 IB of cells as in I. Scale bars: 100 µm. Results in A, D, and G–J are mean ± SEM; *, p < 0.05; **, p < 0.01; ***, p < 0.001. Statistical significance was calculated using Student’s t test (A, D, G–J) or log-rank test (C). See also Figure S3.
Figure 4
Figure 4. p62 Ablation Impairs Cerulein-Induced Neoplastic Progression
(A) H&E staining of pancreatic tissue from indicated 5-week-old mice 2 days after cerulein treatment. Scale bars: 50 µm. (B) H&E staining, amylase and CK19 co-IF, and Sox9 IHC of pancreatic sections from indicated mice 12 days after cerulein treatment. Scale bars: 50 µm. (C) Alcian blue and amylase staining of same tissues as above. Scale bars: 25 µm. (D) Quantification of areas occupied by Alcian blue positive ductal lesions and residual amylase positive acinar cells (n = 6). (E) Ki67 IHC and X-Gal staining of PanIN lesions from cerulein treated mice of indicated genotypes. Scale bars: Ki67, 12.5 µm; X-gal, 25 µm; inset, 12.5 µm. (F) Quantification of Ki67 positive cells in above tissues (n = 6). (G) Quantification of SA-β-gal positive cells (n = 6). (H) Kaplan-Meier survival curves of cerulein or PBS-treated WT mice orthotopically injected with control or p62-deficient KC cells (n = 10). Results in D, F and G are mean ± SEM; *, p < 0.05. Statistical significance was calculated using Student’s t test (D, F, G) or log-rank test (H). See also Figure S4.
Figure 5
Figure 5. NRF2 Mediates Pancreatitis-Accelerated Neoplastic Progression and Induction of Stemness Genes
(A) Quantification of ADM and PanIN lesions in pancreata of 5-week-old mice of indicated genotypes (n = 9). (B) H&E staining of above tissues. Insets show representative PanIN3 in NRF2-expressing tissue and PanIN1 in NRF2-deficient tissue. Scale bars: 50 µm; inset: 25 µm. (C) Kaplan-Meier survival curves of indicated mouse strains (n = 10). (D) IB analysis of pancreatic lysates from above mice. (E, F) Q-RT-PCR analysis of RNA isolated from acinar cells that were cultured for 3 days in Matrigel after transfection with empty or p62 expression vectors (3 µg; n = 3). KrasG12D (E). KrasG12D ;Nrf2−/− (F). (G) Sphere formation by control and NRF2-ablated MIA PaCa-2 and Capan-2 cells with or without NICD1 overexpression. (H) Quantification of SA-β-gal staining of cells as in G. Results in A, E–H are mean ± SEM; *, p < 0.05; **, p < 0.01; ***, p < 0.001. Statistical significance was calculated using Student’s t test (A, E–H) or log-rank test (C). See also Figure S5.
Figure 6
Figure 6. p53 inhibits p62 Induction of Stem/Progenitor Marker Genes
(A, B) Q-RT-PCR mRNA analysis primary KrasG12D acinar cells transfected with control or p62 expression vectors with or without p53 (A) or with or without Nutlin-3 (10 µM) treatment (B) (n = 3 each panel). (C) Quantification of duct-like structures formed by KrasG12D primary acinar cells transfected with either empty or p62 and/or p53 expression vectors and cultured for 4 days in Matrigel with or without Nutlin-3 (n = 3). (D) H&E stained pancreatic sections from mice of indicated genotypes treated with DMSO or Nutlin-3 for 21 days (KrasG12D and cerulein-treated KrasG12D) or 14 days (KrasG12D;IkkαΔpan) (n = 7). (E, F) Pancreata of 5-week-old KrasG12D and KrasG12D;IkkαΔpan mice (n = 7 each group), treated as indicated. Pancreas weight (E) and ADM and PanIN lesions (F). (G, H) Sphere formation by Nutlin-3 (10 µM) or DMSO-treated control or Ikkα-ablated KC (G) and KPC (H) cells with or without p62 or NRF2 ablation. (I) Sphere formation of control or IKKα-ablated Capan-2 human PDAC cells with or without p53 overexpression and/or DAPT treatment (10 µM). Results in A–C, E–I are mean ± SEM; *, p < 0.05; **, p < 0.01; ***, p < 0.001 by Student’s t test. See also Figure S6.
Figure 7
Figure 7. The p62-NRF2-MDM2 Module Controls Stemness and Senescence via Notch Signaling
(A) Effects of p53, p62 and NRF2 overexpression, NRF2 silencing and sulforaphane on Mdm2 promoter activity in transfected KPC cells (n = 3). (B) Chromatin immunoprecipitation assays probing NRF2 and small MAF protein recruitment to the MDM2 promoter in WT and NRF2 ablated MIA PaCa-2 (n = 3) cells. (C) Sphere formation of control or MDM2-ablated MIA PaCa-2 and Capan-2 cells with or without NICD1 overexpression. (D) SA-β-gal staining and γ-H2AX IB of cells as in C. (E) Sphere formation of control and p62-ablated MIA PaCa-2 and Capan-2 cells with or without MDM2 CRISPR-mediated activation vector (n = 3). (F) SA-β-gal staining and γ-H2AX IB of cells as in E. (G) Sphere formation of control and NRF2-ablated MIA PaCa-2 and Capan-2 cells with or without MDM2 CRISPR-mediated activation vector (n = 3). (H) SA-β-gal staining and γ-H2AX IB of cells as in G. (I) A scheme explaining how p62 accumulation, operating via the NRF2-MDM2 module leads to dedifferentiation and cell cycle progression in preneoplastic lesions in pancreas. Results are mean ± SEM; *, p < 0.05; **, p < 0.01; ***, p < 0.001 by Student’s t test. See also Figure S7.

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