Abstract B04: The long noncoding RNA - PRINS as a novel recurrence biomarker and tumor suppressor for adrenocortical carcinoma
Authors: Glover Anthony R. , Zhao Jing T. , Joo Lauren J S , Gill Anthony J. , Robinson Bruce G. , Sidhu Stan B. 
Published: May 2016

Background & Aims: Adrenocortical carcinoma (ACC) has recurrence rates of ~50% and new methods of predicting recurrence are required to complement existing biomarkers (Ki-67 staining) to tailor adjuvant therapy. We have shown expression of PRINS, a long noncoding RNA (lncRNA) can predict ACC recurrence, which is the first clinical association of this lncRNA with outcome in cancer.(1) In keratinocytes where PRINS was discovered, PRINS interacts with the protein nucleophosphmin (NPM) allowing its translocation from the cytoplasm to the nucleus.(2) In the nucleus NPM binds to TP53 to increase its transcriptional activity.(3) TP53 mutations are a key driver of ACC. The aim of this study was to establish a biological basis for PRINS as a biomarker by investigating a tumour suppressor RNA action in ACC.

Method: PRINS expression was quantified in clinical samples using RT-qPCR and Ki-67 staining using immunohistochemistry (IHC). PRINS expression in ACC cells (NCI-H295R) was restored using the mammalian expression vector containing full length of PRINS cDNA transcript (pcDNA3.1[PRINS]). Cell phenotypes were compared to cells transfected with an empty vector (pcDNA3.1[Blank]). Transcriptome analysis was performed on clinical samples of ACC (n=10) and normal adrenal cortex (n=6) using the ArrayStar Human LncRNA V3.0 microarray.

Results: Using RT-PCR NCI-H295R cells were found to have low expression of PRINS compared to clinical samples of normal adrenal cortex (Fold change 0.37, P&lt;0.05) and ACC (Fold change 0.51, P&lt;0.05). Transfection with pcDNA3.1[PRINS] lead to restoration of PRINS expression above that of normal adrenal cortex (P&lt;0.05). In the pcDNA3.1[PRINS] NCI-H295R cells, rates of apoptosis were increased by 49.8% as assessed by Annexin V assays (P&lt;0.05) and cPARP protein expression compared to pcDNA3.1[Blank] transfected cells. Reduced cell proliferation of 30-40% was also found using MTS assays (P&lt;0.05) in PRINS transfected cells. Using RNA FISH, PRINS was localised to the cell cytoplasm. Correlation of ~26,000 mRNA transcripts found PRINS expression to be correlated with 48 mRNAs (Pearson Correlation Coefficients &gt; 80%, corrected P&lt;0.05), including p53 related genes – TNFRSF11B, MKI-67 and TTK. MKI67 codes for the protein Ki-67 and in clinical samples, PRINS relative expression had a moderate negative linear relationship to Ki-67 IHC scoring (n=14, R2=0.38, P=0.02). Following PRINS restoration in NCI-H295R cells, MKI-67 was unchanged, however TP53 was increased (Fold change 1.5, P&lt;0.001) and TNFRSF11B reduced (Fold change 0.56, P&lt;0.05) supporting a mechanism of increased apoptosis.

Conclusions: This first report of PRINS acting as a tumour suppressor in cancer supports the association with ACC outcomes. As PRINS is also under-expressed in breast and prostate cancer it offers potential as a biomarker in other cancers to improve quality of life and further opportunities for research into a novel mechanism of TP53 action in cancer.

This project was supported by a Sydney Vital Research Scholarship Grant.

1. Glover, A. R. et al. Long noncoding RNA profiles of adrenocortical cancer can be used to predict recurrence. Endocrine-Related Cancer 22, 99–109 (2014).

2. Szegedi, K. et al. Expression and Functional Studies on the Noncoding RNA, PRINS. Int J Mol Sci 14, 205–225 (2011).

3. Colombo, E., Marine, J.-C., Danovi, D., Falini, B. & Pelicci, P. G. Nucleophosmin regulates the stability and transcriptional activity of p53. Nature Cell Biology 4, 529–533 (2002).

Citation Format: Anthony R. Glover, Jing T. Zhao, Lauren J S Joo, Anthony J. Gill, Bruce G. Robinson, Stan B. Sidhu. The long noncoding RNA - PRINS as a novel recurrence biomarker and tumor suppressor for adrenocortical carcinoma. [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer: Mechanisms to Medicines ; 2015 Dec 4-7; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2016;76(6 Suppl):Abstract nr B04.