The Tumor Suppressor ARF Can Promote Invasion in the Absence of p53 Activity
Brendan Doyle, Ee Hong Tan, Paul Timpson, Laura M Machesky, Rachel R Ridgway, Rosemary R Jeffery, Richard Poulsom, Jennifer P Morton, Owen J Sansom. Trinity College, Dublin, Ireland; Beatson Institute for Cancer Research, Glasgow, United Kingdom; Cancer Research UK London Research Institute, London, United Kingdom
Background: p53 is mutated in ∼50% of cancers. In this study we combine p53 and APC mutations to develop a novel animal model of colorectal cancer (CRC). Furthermore, we show that the invasive activity of these tumors is due, at least in part, to upregulation of the oncogene Myc and also ARF, a gene more commonly known as a tumor suppressor.
Design: Mice expressing conditional APC knockout (APCfl/fl) were crossed to mice expressing conditional p53 knockout (p53fl/fl) to yield animals heterozygous for APC and homozygous knockout for p53 (APCfl/+ p53fl/fl). By using conditional knockouts the mutations were expressed specifically in the intestine and liver.
Results: APCfl/+ p53fl/fl mice were compared to mice heterozygous for p53 (APCfl/+ p53fl/+) and p53 wild type mice (APCfl/+ p53+/+) both in terms of survival and the development of invasive carcinoma. There was a significant difference in survival between the 3 groups (p=0.02). Moreover, there was a massive difference in the rate of invasive carcinoma across the 3 groups with 79% in the APCfl/+ p53fl/fl group compared with 25% and 29% in the APCfl/+ p53fl/+ and APCfl/+ p53+/+ groups (p=0.004).
We examined the tumors by histology and immunohistochemistry (IHC). The tumors closely recapitulated human CRC, with local invasion and lymph node metastasis in the APCfl/+p53fl/fl group. IHC for ß-catenin showed increased nuclear ß-catenin at the invasive front of tumors, this was mirrored by increased Myc, which is downstream of ß-catenin. Interestingly, ARF was also found to be increased at the invasive front. ARF is normally induced in response to oncogenic stress (including increased Myc) and stabilises p53. To determine if this increase in ARF was functional we overexpressed ARF in p53 null cells. An invasion assay showed that p53 null cells overexpressing ARF invaded significantly deeper than controls (p<0.001).
To further confirm these findings we crossed our experimental mice to Myc (Mycfl/+) and ARF (ARFfl/fl) knockout mice. In keeping with our findings there was a significant decrease in the rate of invasive tumors in the APCfl/+p53fl/fl Mycfl/+ and APCfl/+p53fl/fl ARFfl/fl groups compared to controls.
Conclusions: This study shows that in a p53 null setting invasion is driven, at least in part, by the oncogene Myc and surprisingly by the tumor suppressor ARF. Although these experiments have been done in a model of CRC, the fact that p53 mutations are common across a wide spectrum of tumors makes these potentially attractive therapeutic targets.
Monday, March 19, 2012 11:45 AM
Platform Session: Section G2, Monday Morning