Haploinsufficiency of p18(INK4c) sensitizes mice to carcinogen-induced tumorigenesis

Abstract

The INK4 family of cyclin-dependent kinase (CDK) inhibitors negatively regulates cyclin D-dependent CDK4 and CDK6 and thereby retains the growth-suppressive function of Rb family proteins. Mutations in the CDK4 gene conferring INK4 resistance are associated with familial and sporadic melanoma in humans and result in a wide spectrum of tumors in mice. Whereas loss of function of other INK4 genes in mice leads to little or no tumor development, targeted deletion of p18(INK4c) causes spontaneous pituitary tumors and lymphoma late in life. Here we show that treatment of p18 null and heterozygous mice with a chemical carcinogen resulted in tumor development at an accelerated rate. The remaining wild-type allele of p18 was neither mutated nor silenced in tumors derived from heterozygotes. Hence, p18 is a haploinsufficient tumor suppressor in mice.

FIG. 1.

Tumor-free survival in untreated wild-type and p18 mutant mice and in mice exposed to DMN. p18^+/+, p18^+/−, and p18^−/− mice were either untreated or fed with DMN (0.0005%) in their drinking water continuously from the age of 8 weeks. Statistically significant differences in survival were detected between cohorts of DMN-treated p18^−/− and DMN-treated p18^+/− mice (P < 0.001) and between DMN-treated p18^+/− and DMN-treated p18^+/+ mice (P < 0.05).

FIG. 2.

DMN treatment accelerated pituitary tumorigenesis in p18-deficient mice. Pituitary glands (pointed by arrows) from p18^+/+, p18^+/−, and p18^−/− mice, either untreated or exposed to DMN, were microscopically examined at 6 months (A to L) and 9 months (M to P) of age either directly (top two rows; bars, 1 mm) or after hematoxylin and eosin (H/E) staining (lower two rows; bars, 200 μm). WT, wild type. Anterior lobe (A), intermediate lobe (I), neurohypophysis (N), and tumor (T) are indicated.

FIG. 3.

Mutation in p18 gene sensitized mice to DMN-induced lung tumors. (A to C) The rate of lung tumor development varied between age-matched (11 months) p18^+/+, p18^+/−, and p18^−/− mice after DMN exposure. Gross alveolar and bronchiolar adenomas (white spots) are identified by arrows. Note that p18 mutant lungs developed more tumors. (D to F) Alveolar or bronchiolar origin of lung tumor development. Lungs of DMN-treated mice at different ages were microscopically examined after hematoxylin-eaosin staining and exhibited alveolar or bronchiolar hyperplasia (D), papillary alveolar or bronchiolar adenoma with mucinous cell differentiation (E), and alveolar or bronchiolar carcinoma (F). The inset in panel F is a higher magnification showing tumor infiltration of adjacent pulmonary parenchyma. (G) The number of lung tumors that developed in each mouse exposed to DMN increases with p18 gene mutation. A statistically significant difference in tumor multiplicity was seen in mice of a different p18 genotype following exposure to DMN. WT, wild type. (H) The average size of DMN-induced lung tumors was larger in p18 mutant mice than in wild-type mice.

FIG. 4.

Mutation in the p18 gene sensitized mice to DMN-induced liver tumors. (A to F) Livers from p18^−/−, p18^+/−, and p18^+/+ mice exposed to DMN were examined at the ages of 12 weeks (A to C) and 34 weeks (D to F). Normal gross liver architecture was retained in DMN-treated mutant mice at a young age despite the development of multiple hemangiosarcomas (red spots pointed by the arrows), but was not observed in p18 null mice after 34 weeks of age. WT, wild type. (G) Microscopic examination of hemangiosarcoma that developed in the liver of a 40-week-old p18^−/− mouse exposed to DMN. Note that irregular vascular spaces have destroyed the normal hepatic parenchyma. (H) Higher magnification of the boxed area in (G) showing proliferating malignant endothelial cells replacing normal hepatic parenchyma.

FIG. 5.

p18 is a haploinsufficient tumor suppressor. (A and B) Retention of the remaining wild-type p18 allele in liver (A) and lung (B) tumors from wild-type and p18 heterozygous mice. Genomic DNA was extracted from microdissected tumor cells or tails of p18^+/− or p18^−/− mice and subjected to PCR amplification with two pairs of primers flanking either the wild-type (WT) or mutated (mt) allele of the p18 gene. PCR products were resolved by agarose gel (1.2%) electrophoresis. The entire coding regions of p18 from liver tumor samples 2 to 11 and from lung tumor samples 2 to 8 were sequenced, and no mutation was found. (C to J) The expression of p18^INK4c protein in three different types of tumors (liver hemangiosarcoma, lung adenoma, and abdominal sarcoma) from mice of three different p18 genotypes was determined by immunohistological staining with an affinity-purified polyclonal antibody specific to p18^INK4c protein (8). Bars in all panels are 50 μm.