Loss of 9p leads to p16(INK4A) down-regulation and enables RB/E2F1-dependent cell cycle promotion in gastrointestinal stromal tumours (GISTs)

Loss of chromosome 9p is a reliable predictor of malignant behaviour in gastrointestinal stromal tumours (GISTS). p16(INK4A) located at 9p21 inhibits the CDK4/6/cyclin D complex from phosphorylating RB. Phosphorylation of RB through CDK4/6/cyclin D in early G(1) phase frees the transcription factor E2F1 from RB and enables mRNA transcription of genes essential for G(1)/S phase transition. This study aims to determine the impact of 9p loss on mRNA and protein expression of p16(INK4A) and further key cell cycle regulators in the different phases of the cell cycle. Sixty primary GISTS previously characterized for 9p loss by comparative genomic hybridization were analysed for mRNA expression of p16(INK4A), p15(INK4B), CDK4, CDK6, cyclin D, p 21(CIP1)p27(KIP1), CDK2, cyclin E, cyclin B, RB and E2F1, using quantitative RT-PCR. The protein expression of CDK6, CDK2, p2(CIP1), p27(KIP1) and phosphorylated RB (S807/S811) was evaluated using protein arrays as a novel and highly sensitive platform for profiling of protein abundance and protein phosphorylation. In parallel, the nuclear percentages of immunohistochemical staining for p16(INK4A), cyclin D, E2F1 and RB were quantified on a tissue microarray. GISTS with 9p loss had significantly higher proliferation rates, higher metastatic behaviour and shorter disease-free survival. On the molecular level, GISTS with 9p loss had a significantly reduced mRNA as well as nuclear protein expression of p16(INK4A). RB was significantly more phosphorylated in these tumours, together with increased mRNA expression and nuclear staining for E2F1 Furthermore, GISTS with 9p loss had up-regulation of the late G(1)/S phase promoters CDK2 and cyclin E. We conclude that loss of 9p accompanied by early G(1) phase inhibitor p16(INK4A) down-regulation in GISTS facilitates phosphorylation of RB, enabling F2F1-dependent transcription of genes essential for late G(1)/S phase transition. This study provides a possible basis for the accelerated proliferation and particularly malignant behaviour in GISTS with 9p loss. Copyright (C) 2008 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.