p19Arf suppresses growth, progression, and metastasis of Hras-driven carcinomas through p53-dependent and -independent pathways

Abstract

Ectopic expression of oncogenes such as Ras induces expression of p19(Arf), which, in turn, activates p53 and growth arrest. Here, we used a multistage model of squamous cell carcinoma development to investigate the functional interactions between Ras, p19(Arf), and p53 during tumor progression in the mouse. Skin tumors were induced in wild-type, p19(Arf)-deficient, and p53-deficient mice using the DMBA/TPA two-step protocol. Activating mutations in Hras were detected in all papillomas and carcinomas examined, regardless of genotype. Relative to wild-type mice, the growth rate of papillomas was greater in p19(Arf)-deficient mice, and reduced in p53-deficient mice. Malignant conversion of papillomas to squamous cell carcinomas, as well as metastasis to lymph nodes and lungs, was markedly accelerated in both p19 (Arf)- and p53-deficient mice. Thus, p19(Arf) inhibits the growth rate of tumors in a p53-independent manner. Through its regulation of p53, p19(Arf) also suppresses malignant conversion and metastasis. p53 expression was upregulated in papillomas from wild-type but not p19( Arf)-null mice, and p53 mutations were more frequently seen in wild-type than in p19( Arf)-null carcinomas. This indicates that selection for p53 mutations is a direct result of signaling from the initiating oncogenic lesion, Hras, acting through p19(Arf).

Figure 1. Skin Tumor Multiplicity, Size, and Progression in p19^Arf-Deficient Mice

(A) Average number of papillomas (more than 2 mm in diameter) per mouse is plotted versus the number of weeks postinitiation. Both p19^Arf (Arf)^+/− and p19^Arf−/− mice show greater numbers of tumors than p19^Arf+/+ mice. (B) Comparison of papilloma size (in mm) between p19^Arf+/+, p19^Arf+/−, and p19^Arf−/− mice through 28 wk postinitiation. An increase in the largest size class of tumors is seen in p19^Arf+/− and p19^Arf−/− mice but not p19^Arf+/+ mice. (C) Percentage of mice bearing at least one carcinoma is plotted versus the number of weeks postinitiation. p19^Arf−/− mice show the shortest latency and greatest incidence of carcinoma conversion, with p19^Arf+/− mice showing an incidence between the p19^Arf−/− and p19^Arf+/+ mice. Time of appearance of lymph node metastasis is noted above the graph as a vertical line for each mouse analyzed. Metastasis to lymph node occurred frequently and sooner in p19^Arf-deficient mice than in wild-type mice.

Figure 2. Metastasis of Primary SCC to Lymph Nodes and Lungs in p19^Arf-Deficient Mice

(A) Underside of skin from tumor-bearing mouse shows newly formed blood vessels surrounding tumor site (arrow) and leading to inguinal lymph node (arrowhead). (B) Enlarged inquinal lymph node (left) containing metastatic SCC and blood vessel formation (arrow) compared to normal lymph node (right). (C) H&E stain of carcinoma section with prominent blood vessel (bv). Carcinoma cells (ca) have penetrated blood vessel wall (arrow). (D) H&E stain of lymph node bearing infiltrating SCC cells (arrow) among normal lymphocytes (arrowhead). (E) H&E stain of lymph node bearing metastatic differentiated SCC. (F) Immunostain with pan-keratin antibody of papilloma. (G) Immunostain with pan-keratin antibody of lymph node with metastatic SCC. (H and I) H&E stain of normal lung (arrowhead) with large metastatic SCC deposit (arrow). (J) H&E stain of lung metastasis with secondary site of infiltration (arrow). (D–G, J): 20× magnification. Inserts in (E–G): 40× magnification.

Figure 3. Reduced p53 Expression in Skin Tumors from p19^Arf-Deficient Mice

(A) Western blot analysis of nuclear lysates from skin tumors from p19^Arf (Arf)^+/+, p19^Arf+/−, and p19^Arf−/− mice using p53-specific antibody. PA, papilloma; skin IR, irradiated normal skin (B) p53 immunostain of paraffin-embedded skin tumor sections from p19^Arf+/+, p19^Arf+/−, and p19^Arf−/− mice (arrows indicate positive stained cells) (top). p53 immunostain of irradiated papillomas (IR) from p19^Arf+/+ and p19^Arf−/− mice (bottom). p53 is not detected in normal skin or tumors from p19^Arf−/− mice, but is induced by irradiation in both normal and tumor cells from p19^Arf−/− mice.

Figure 4. LOH of Wild-Type p19^Arf Allele in p19^Arf+/− Tumors

(A) LOH analysis by semiquantitative PCR of the wild-type p19^Arf allele in p19^Arf+/− papillomas and carcinomas. Gradient made from kidney DNA used for quantitation of wt/mu ratio (top row). wt, wild-type allele; mu, knockout allele; asterisk, loss or reduction of p19^Arf wild-type band. (B) Western blot analysis of nuclear lysates from papillomas (PA) and carcinomas (CA) from p19^Arf+/+, p19^Arf+/−, and p19^Arf−/− mice.

Figure 5. Tumor Multiplicity and Proliferative Index in p19^Arf /p53 Compound Mutant Mice

(A) Average number of papillomas (more than 2 mm in diameter) per mouse is plotted against the number of weeks post-initiation. (B) Image of wild-type, p19^Arf (Arf)^−/−, p53^−/−, and p19^Arf−/−p53^−/− mice with skin tumors at time of sacrifice. Wild-type mice show large exophytic tumors, while both p19^Arf- and p53-deficient mice have endophytic tumors. Note larger tumors in p19^Arf /p53 compound mutant mice relative to p53 single mutants. (C) BrdU-positive cells in papillomas from wild-type, p53^−/−, p19^Arf−/−, and p19^Arf−/−p53^−/− mice at 10 wk postinitiation. (Bars represent average counts ± standard deviation from ten fields and five mice). p53^−/− tumors show significantly fewer BrdU-positive cells than either p19^Arf−/− or wild-type tumors (p < 0.05, Wilcoxon one-sided t-test).

Figure 6. Tumor Progression and p53 LOH in p19^Arf /p53 Compound Mutant Mice

(A) Average number of carcinomas per mouse is plotted against the number of weeks postinitiation. Tumor progression was accelerated in all p19^Arf (Arf)- and p53-deficient genotypes compared to wild-type littermates. Carcinoma latency and multiplicity was almost identical for p19^Arf−/− mice regardless of p53 genotype (p53^+/+, p53^+/−, or p53^−/−). (B) LOH of the wild-type p53 allele by semiquantitative PCR in p19^Arf /p53 compound tumors. Gradient made from kidney DNA used for quantitation of wt/mu ratio (top row). wt, wild-type allele; mu, knockout allele; asterisk, tumors with reduction of wild-type p53.

Figure 7. An Integrated Model of SCC Progression

At the genetic level, treatment of mouse skin with DMBA induces mutations in Hras resulting in initiated cells that express constitutively active Ras protein (grey rectangles). TPA treatment is required for clonal expansion of these Hras mutant cells to form papillomas. Frequent duplication of the mutant Ras allele in papillomas indicates increased Ras signaling is favored. Mutation of p53, as well as additional Ras gene amplification, is seen in carcinomas, particularly in the most aggressive tumors (black rectangles). At the signaling level, mutant Ras upregulates p19^Arf (Arf), which in turn activates p53. p19^Arf, in turn, inhibits growth of Hras-driven tumors in a p53-independent manner. p19^Arf, acting through p53, also inhibits malignant progression and metastasis. As Ras signals through p19^Arf and p53, selection for subsequent mutations in p19^Arf or p53 is a direct consequence of the initial Ras mutation. In this model, Ras drives tumor progression through direct signaling effects and by dictating the evolution pathway of the tumor.