Both p16(Ink4a) and the p19(Arf)-p53 pathway constrain progression of pancreatic adenocarcinoma in the mouse - PubMed (original) (raw)

. 2006 Apr 11;103(15):5947-52.

doi: 10.1073/pnas.0601273103. Epub 2006 Apr 3.

Andrew J Aguirre, Gerald C Chu, Kuang-Hung Cheng, Lyle V Lopez, Aram F Hezel, Bin Feng, Cameron Brennan, Ralph Weissleder, Umar Mahmood, Douglas Hanahan, Mark S Redston, Lynda Chin, Ronald A Depinho

Affiliations

Both p16(Ink4a) and the p19(Arf)-p53 pathway constrain progression of pancreatic adenocarcinoma in the mouse

Nabeel Bardeesy et al. Proc Natl Acad Sci U S A. 2006.

Abstract

Activating KRAS mutations and p16(Ink4a) inactivation are near universal events in human pancreatic ductal adenocarcinoma (PDAC). In mouse models, Kras(G12D) initiates formation of premalignant pancreatic ductal lesions, and loss of either Ink4a/Arf (p16(Ink4a)/p19(Arf)) or p53 enables their malignant progression. As recent mouse modeling studies have suggested a less prominent role for p16(Ink4a) in constraining malignant progression, we sought to assess the pathological and genomic impact of inactivation of p16(Ink4a), p19(Arf), and/or p53 in the Kras(G12D) model. Rapidly progressive PDAC was observed in the setting of homozygous deletion of either p53 or p16(Ink4a), the latter with intact germ-line p53 and p19(Arf) sequences. Additionally, Kras(G12D) in the context of heterozygosity either for p53 plus p16(Ink4a) or for p16(Ink4a)/p19(Arf) produced PDAC with longer latency and greater propensity for distant metastases relative to mice with homozygous deletion of p53 or p16(Ink4a)/p19(Arf). Tumors from the double-heterozygous cohorts showed frequent p16(Ink4a) inactivation and loss of either p53 or p19(Arf). Different genotypes were associated with specific histopathologic characteristics, most notably a trend toward less differentiated features in the homozygous p16(Ink4a)/p19(Arf) mutant model. High-resolution genomic analysis revealed that the tumor suppressor genotype influenced the specific genomic patterns of these tumors and showed overlap in regional chromosomal alterations between murine and human PDAC. Collectively, our results establish that disruptions of p16(Ink4a) and the p19(ARF)-p53 circuit play critical and cooperative roles in PDAC progression, with specific tumor suppressor genotypes provocatively influencing the tumor biological phenotypes and genomic profiles of the resultant tumors.

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Conflict of interest statement

Conflict of interest statement: No conflicts declared.

Figures

Fig. 1.

Fig. 1.

Deficiency in p53 or p16Ink4a cooperates with oncogenic KrasG12D to produce PDAC. (A) Hematoxylin/eosin stain of a PDAC (T, tumor) arising in a p53lox/lox p16+/+ mouse. Note invasion of duodenum (Du). (Scale bar: A and D, 200 μm; I, N, and O, 100 μm; B, C, E–H, and J–L, 50 μm.) (B) High-magnification view of the tumor in A showing features of ductal adenocarcinoma. (C) Tumor from p53 lox/lox p16 −/− mouse showing poorly differentiated adenocarcinoma (yellow arrows) admixed with anaplastic epithelioid cells characterized by giant tumor nuclei (black arrows) and eosinophilic inclusions (arrowheads). (D) Invasive PDAC arising in Pdx1-Cre LSL-KrasG12Dp16Ink4a−/− mouse. (E) High-magnification of D showing ductal adenocarcinoma histology. (F) Another region of the tumor in E showing sarcomatoid differentiation. (G) PDAC from p53 lox/+;p16 Ink4a+/− mouse. (H) Positive staining of the tumor in G for the ductal marker cytokeratin 19. (I) Anaplastic histology in PDAC from the p53 _lox/+;p16+/−_model. (J) Well differentiated p16 Ink4a/p19 Arf lox/+ PDAC. (K) Positive staining of tumor in J for cytokeratin 19. (L) p16 Ink4a/p19 Arf lox/+ tumor showing sarcomatoid histology. (M) p53 lox/+ p16 +/− tumor invading the duodenum. (N and O) p16 Ink4a/p19 Arf lox/+ tumors with metastases to the lung (N) and liver (LV; O).

Fig. 2.

Fig. 2.

Molecular analyses of PDAC cell lines and primary tumor specimens from mice with p53 and p16 mutant animals. (A) PCR reactions to detect the _p53_-WT (+) and p53lox alleles (Upper) and _p53-_null (−) allele (Lower) in normal tissue from p53lox/lox (lane 1) and p53 lox/+ (lane 7) mice and tumor cell lines (lanes 2–6). All tumor cell lines show only the _p53_-null allele. Germ-line p16Ink4a status is indicated at the top. (B) Western blot for p16Ink4a and p19Arf expression in cell lines derived from p53lox/lox mice with various p16Ink4a genotypes. The negative and positive controls are in lanes 9 and 10, respectively. α-Tubulin is shown as a loading control. (C) PCR analysis of the p53+ and p53lox alleles (Upper) and the p53− (/) allele (Lower) demonstrates loss of the p53+ allele in all tumors from _p53_lox/+ p16+/− mice (lanes 2–6). Lane 1 shows the WT control specimen. (D) Western blot analysis shows low or absent p16Ink4a expression in five of six tumor cell lines from p53 lox/+ p16 +/− mice (Right, lanes 1–6), whereas all retain p19Arf expression. Negative controls are in lanes 7 and 12. p16Ink4a expression in PDAC cell lines from p53lox/lox p16 +/+ (lane 8) and p53 lox/lox p16 +/− (lane 10) mice is shown as a reference (Right). (E) PCR analysis of the p16Ink4a− and p16Ink4a+ alleles demonstrates LOH in one of six samples (lane 5). Normal tissue specimens are shown as controls for the p16Ink4a+/+, p16 Ink4a−/−, and p16 Ink4a+/+ alleles (lanes 1–3). (F) Methylation-specific PCR assay to detect methylated CpG islands in the p16Ink4a promoter region reveals hypermethylation in three tumor lines (lanes 1, 4, and 5). Upper and Lower show the methylated (M) and unmethylated (U) alleles, respectively. Negative and positive controls are in lanes 7 and 8.

Fig. 3.

Fig. 3.

Molecular analyses of PDAC cell lines from p16Ink4a/p19Arf lox/+ and p16INK4A/p19Arf+/+ mice. (A) Western blot analysis p16Ink4a and p19Arf expression in lysates from tumor cell lines from p16INK4A/p19Arf lox/+ mice. (B) PCR analysis of p16INK4A/p19Arf alleles in tumor cell lines from p16Ink4a/_p19_Arf lox/+ mice shows only the recombined p16INK4A/p19Arf allele (Left, lanes 2–8). Normal tissue from p16Ink4a/_p19_Arf lox/+ mice shows WT (+) and unrecombined (lox) alleles. Tumor no. 738 (Left, lane 6; Right, lane 2) shows a biallelic deletion of the entire p16Ink4a/p19Arf locus. (C) Western blot analyses of γ-irradiated tumor cell lines (+) shows induction of total p53 protein levels and phosphorylation of p53 on Ser-15, consistent with present and functional p53 protein. −, untreated. Cdk4 protein levels are shown as a loading control. (D) Western blot analysis shows absence of p16Ink4a and p19Arf expression in Pdx1-Cre LSL-KrasG12D cell lines (lanes 1 and 2). Lanes 3 and 4 show positive and negative controls. (E) PCR analysis for the presence of p16Ink4a/p19Arf exon 2, p16Ink4a exon 1, and cytokeratin 19 sequences in cell lines from Pdx1-Cre LSL-KrasG12D mice (lanes 2 and 3) demonstrates biallelic deletion of p16Ink4a/p19Arf. Lane 1 shows normal control DNA.

Fig. 4.

Fig. 4.

aCGH analysis of mouse PDAC. (A) Hierarchical clustering of aCGH profiles reveals three distinct clusters. Sample names are given above the profile followed by an underscore and the genotype class (i.e., “106_6” indicates the profile for tumor no. 106, which is in genotype class 6. The genotype classes are: 1, p53 lox/lox;p16 Ink4a +/+; 2, p53 lox/lox;p16 Ink4a +/−; 3, p53 lox/lox;p16 Ink4a −/−; 4, p53 lox/+;p16 Ink4a +/−; 5, p16 Ink4a/p19Arf lox/+; 6, p16INK4A/p19Arf lox/lox. (B and C) Focal amplifications of Kras2 in p16Ink4a/_p19Arf_-deficient (B) and Myc in _p53_-deficient (C) tumors are shown. Minimal common regions of amplification are shown by blue lines.

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