Mechanistic Investigation of the p16[INK4A] Pathway
L Kehoe, CD Spillane, M Gallagher, O Sheils, C Martin, JJ O'Leary. Trinity College Dublin, Dublin, Ireland; Coombe Women and Infants University Hospital, Dublin, Ireland
Background: Unravelling effects of high risk HPV oncoproteins has led to the discovery of dysregulated proteins and potential biomarkers. One of these is p16[INK4A]. Although p16 protein expression is used diagnostically, little is known about its transcriptional regulation and cell cycle involvement.
Design: Using HaCaT, a p16 protein null cell line, we aimed to determine cause and effects of p16 up regulation in cervical cancer (CC). We also examined the expression profile of a naturally occurring transcript [ANRIL] in CC cell lines, CIN, cGIN squamous cell carcinoma (SCC) and adenocarcinoma.
Results: HaCaT cells have been reported p16 null due to promoter methylation. We have discovered HaCaTs are expressing p16 mRNA and its promoter is unmethylated. Western blots indicate lack of detectable protein expression. We hypothesise p16 is undergoing post transcriptional regulation (PTR) in HaCaT cells. This is possibly due to interference by RNA molecules such as natural anti-sense transcripts (NATs) and microRNAs (miRNA). Results show the presence of p16 message at the polysome. Though miR-24 has been described in PTR of p16, we did not find this effect in HaCaT cells. A large NAT [ANRIL], which overlaps p14/p15, may play a role in regulating the INK4A/ARF locus. This NAT can be detected in total and polysomal RNA from HaCaT, HeLa and C33A cells. Due to its large size, different regions were investigated. All were present in cell lines, indicating further processing of any of these regions to small RNAs is not a factor in p16 PTR in HaCaT. Evaluation of ANRIL in CC and pre-cancer found no significance on comparison to normal cervix in CIN1, CIN3 and adenocarcinoma. Over-expression of ANRIL was found in cGIN and SCC cases. This data indicates expression of the INK4A/ARF locus and its NAT [ANRIL] is uncoupled in CC. Comparative miRNA profiling of HaCaT p16 protein incompetent cells to p16 competent cell lines [both HPV positive and negative] revealed a panel of 26 miRNAs; possible regulators of p16 transcript.
Conclusions: Bypassing p16 cellular control is an important mechanism in cancer development. We have found p16 control in HaCaT cells is bypassed by preventing p16 protein expression, facilitating creation of the HaCaT immortal but non-tumourgenic phenotype. We have also shown expression of ANRIL and the INK4A/ARF locus is uncoupled in CC, indicating HPV induced p16 over-expression is autonomous in CC and pre-cancer confirming the dominant role of HPV E7 in this setting.
Wednesday, March 24, 2010 9:30 AM
Poster Session V # 232, Wednesday Morning