Chimeras of p14ARF and p16: Functional Hybrids with the Ability to Arrest Growth

Richard T. Williams,Lisa M. Barnhill,Mitchell B. Diccianni,Wen-Der Lin,Alice L. Yu,Ayse Batova,Huan-Hsien Kuo,Carl G. Maki

doi:10.1371/journal.pone.0088219

Richard T. Williams, Lisa M. Barnhill + Show 6 more

Open Access

https://doi.org/10.1371/journal.pone.0088219

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Journal: PloS one	Publication Date: Feb 5, 2014
Citations: 36	License type: CC0 1.0

Affiliation: University of California, San Diego

Abstract

The INK4A locus codes for two independent tumor suppressors, p14ARF and p16/CDKN2A, and is frequently mutated in many cancers. Here we report a novel deletion/substitution from CC to T in the shared exon 2 of p14ARF/p16 in a melanoma cell line. This mutation aligns the reading frames of p14ARF and p16 mid-transcript, producing one protein which is half p14ARF and half p16, chimera ARF (chARF), and another which is half p16 and half non-p14ARF/non-p16 amino acids, p16-Alternate Carboxyl Terminal (p16-ACT). In an effort to understand the cellular impact of this novel mutation and others like it, we expressed the two protein products in a tumor cell line and analyzed common p14ARF and p16 pathways, including the p53/p21 and CDK4/cyclin D1 pathways, as well as the influence of the two proteins on growth and the cell cycle. We report that chARF mimicked wild-type p14ARF by inducing the p53/p21 pathway, inhibiting cell growth through G2/M arrest and maintaining a certain percentage of cells in G1 during nocodazole-induced G2 arrest. chARF also demonstrated p16 activity by binding CDK4. However, rather than preventing cyclin D1 from binding CDK4, chARF stabilized this interaction through p21 which bound CDK4. p16-ACT had no p16-related function as it was unable to inhibit cyclin D1/CDK4 complex formation and was unable to arrest the cell cycle, though it did inhibit colony formation. We conclude that these novel chimeric proteins, which are very similar to predicted p16/p14ARF chimeric proteins found in other primary cancers, result in maintained p14ARF-p53-p21 signaling while p16-dependent CDK4 inhibition is lost.

Highlights

The transcript driven off the p16 promoter coded for the first 80 amino acids of p16 followed by 64 non-p16/non-p14ARF amino acids (p16-Alternative Carboxyl Terminus [p16Alternate Carboxyl Terminal (p16-ACT)]) (Fig. 1C)
Mutations predicted to create p16-14ARF chimeric proteins have been documented in many primary cancers [11] and [24] including familial melanoma [25], squamous cell carcinoma of the skin [26] and squamous cell carcinoma of the esophagus [13] and such mutations represent an important avenue of cancer research
We report a unique 1 bp deletion, 1 bp substitution in exon 2 of p16/p14ARF found in a melanoma cell line which creates two unique p16/p14ARF chimeras

Summary

Introduction

P16 (CDKN2A) and p14ARF are two overlapping genes in the INK4A locus located on chromosome 9p21 They share common exons 2 and 3 yet have different first exons, 1a for p16 and 1b for p14ARF, which are 20 kb apart and under the control of independent promoters. P14ARF, on the other hand, prevents p53 degradation by sequestering the E3 ubiquitin-protein ligase mouse double minute 2 homolog (MDM2) This stabilization of p53 leads to the induction of the cyclin-dependent kinase inhibitor p21 (CDKN1A) and subsequently halts cell growth by arresting cells in the G1 and G2/M phases of the cell cycle. We assessed the impact of these proteins on cell cycle regulation and growth suppression to understand their potential roles in cancer progression

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