Abstract
In tumors with WT p53, alternate mechanisms of p53 inactivation are reported. Here, we have identified a long noncoding RNA, PITAR (p53 Inactivating TRIM28 associated RNA), as an inhibitor of p53. PITAR is an oncogenic Cancer/testis lncRNA and is highly expressed in glioblastoma (GBM) and glioma stem-like cells (GSC). We establish that TRIM28 mRNA, which encodes a p53-specific E3 ubiquitin ligase, is a direct target of PITAR. PITAR interaction with TRIM28 RNA stabilized TRIM28 mRNA, which resulted in increased TRIM28 protein levels and reduced p53 steady-state levels due to enhanced p53 ubiquitination. DNA damage activated PITAR, in addition to p53, in a p53-independent manner, thus creating an incoherent feedforward loop to inhibit the DNA damage response by p53. While PITAR silencing inhibited the growth of WT p53 containing GSCs in vitro and reduced glioma tumor growth in vivo, its overexpression enhanced the tumor growth in a TRIM28-dependent manner and promoted resistance to Temozolomide. Thus, we establish an alternate way of p53 inactivation by PITAR, which maintains low p53 levels in normal cells and attenuates the DNA damage response by p53. Finally, we propose PITAR as a potential GBM therapeutic target.
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
Conflict of Interest: The authors declare no potential conflicts of interest.
Abbreviations: LncRNA-Long noncoding RNA, PITAR-p53 Inactivating TRIM28 associated RNA, TRIM28-Tripartite Motif Containing 28, CSC-Cancer stem cells, GBM-Glioblastoma, GSC-Glioma stem cells
We have made several additions in the form of new experiments to address all concerns raised both reviewers and are described below: 1) Two siRNAs are used (Figure 2, 5 and supplementary Figure 2). 2) A better quality p53 ubiquitination western blot is provided (Figure 5F). 3) We show that PITAR overexpression is unable to inhibit p53 in TRIM28 silenced conditions (Figure 5K, L, M, N and supplementary Figure 5F and G) 4) The impact of PITAR silencing and overexpression on p53 is shown in two cell lines (supplementary Figure 5) 5) We show that the tumor growth-promoting functions of overexpressed PITAR is compromised in TRIM28 silenced cells(Figure 7H, I and supplementary Figure 9C). 6) We established a firm connection between DNA damage induced PITAR (in a p53 independent manner) and DNA damage response of p53. Essentially, we show that DNA-damage response of p53 is enhanced in PITAR silenced conditionsdue to the fact the TRIM28 mediated degradation of p53 will be absent (supplementary Figure 8E, F, G and H).
