PITAR, a DNA damage-inducible Cancer/Testis long noncoding RNA, inactivates p53 by binding and stabilizing TRIM28 mRNA

Reviewer #1 (Public Review):

The authors characterized a new non-coding RNA, which they named as PITAR. They first showed that the PITAR expression levels are higher in glioblastoma, and then demonstrated that knockdown of PITAR in glioblastoma cells decreased cell growth, induced G0/G1 arrest and apoptosis. They further identified the E3 ubiquitin ligase TRIM28 is the target of PITAR, and showed that PITAR bound to the TRIM28 mRNA and regulated the stability and expression of the latter. Since TRIM28 has been reported to be an E3 ubiquitin ligase for the tumor suppressor p53, the authors tried to link the PITAR function to p53 regulation. They showed that one PITAR siRNA increased the levels of p53 and p21, and the stability of p53, and these effects could be diminished by overexpression of TRIM28. They also showed that PITAR overexpression decreased the levels of adriamycin-induced p53/p21 expression and reversed DNA damage-induced G2/M arrest. Lastly, the authors showed that PITAR siRNA decreased the growth of glioblastoma, while PITAR overexpression increased glioblastoma growth and counteracted temozolomide for its anti-glioblastoma activity.

Overall, the manuscript has provided preliminary evidence supporting the important role of PITAR in the regulation of the growth and drug resistance of glioblastoma. The results supporting the regulation of PITAR on TRIM28 appear to be convincing. However, the study suffers significant weaknesses summarized as below.

(1) Only one PITAR siRNA was tested in majority of the experiments, which compromises the validity of the results. Some results are inconsistent. For example, Fig 2G indicates that PITAR siRNA caused G1 arrest. However, PITAR overexpression in the same cell line did not show any effect on cell cycle progression in Fig 5I.

(2) The conclusion that PITAR inactivates p53 through regulating TRIM28, which is highlighted in the title of the manuscript, is not supported by convincing results. Although the authors showed that a PITAR siRNA increased while PITAR overexpression decreased p53 level, the siRNA only marginally increased the stability of p53 (Fig 5E). The p53 ubiquitination level was barely affected by PITAR overexpression in Fig 5F. To convincingly demonstrate that PITAR regulates p53 through TRIM28, the authors need to show that this regulation is impaired/compromised in TRIM28-knockout conditions. The authors only showed that TRIM28 overexpression suppressed PITAR siRNA-induced increase of p53, which is not sufficient. Note that only one cell line was investigated in Fig 5.

(3) Another major weakness of this manuscript is that the authors did not provide any evidence indicating that the glioblastoma-promoting activities of PITAR were mediated by its regulation of p53 or TRIM28 (Fig 6 and Fig 7). Thus, the regulation of glioblastoma growth and the regulation of TRIM28/p53 appear to be disconnected.

(4) It is not clear what kind of message the authors tried to deliver in Fig 7F/G. Based on the authors' hypothesis, DNA damaging agents like TMZ would induce PITAR to inactivate p53, which would compromise TMZ's anti-cancer activity. However, the data show that TMZ was very effective in the inhibition of U87 growth. The authors may need to test whether PITAR downregulation, which would increase p53 activity, have any effects on TMZ-insensitive tumors. Such results are more therapeutically relevant.

(5) Lastly, the model presented in Fig 7H is confusing. It is not clear what the exact role of PITAR in the DNA damage response based on this model. If DNA damage would induce PITAR expression, this would lead to inactivation of p53 as revealed by this manuscript. However, DNA damage is known to activate p53. Do the authors want to imply that PITAR induction by DNA damage would help to bring down the p53 level at the end of DNA damage response? The presented data do not support this role unfortunately.

Reviewer #2 (Public Review):

This manuscript identified a long noncoding RNA, PITAR (p53 Inactivating TRIM28 associated RNA), as an inhibitor of p53. PITAR is highly expressed in glioblastoma (GBM) and glioma stem-like cells (GSC). The authors found 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, enhanced p53 ubiquitination, and attenuated DNA damage response. 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 and promoted resistance to Temozolomide. DNA damage also activated PITAR, in addition to p53, thus creating an incoherent feedforward loop. Together, this study established an alternate way of p53 inactivation and proposed PITAR as a potential therapeutic target.

P53 is a well-established tumor suppressor gene contributing to cancer progression in many human cancers. It plays a vital role in preserving genome integrity and inhibiting malignant transformation. p53 is mutated in more than 50% of human cancers. In cancers that do not carry mutations in p53, the inactivation occurs through other genetic or epigenetic alterations. Therefore, further study of the mechanism of regulation of wt-p53 remains vital in cancer research. This study identified a novel LncRNA PITAR, which is highly expressed in glioblastoma (GBM) and glioma stem-like cells (GSCs) and interacts with and stabilizes TRIM28 mRNA, which encodes a p53-specific E3 ubiquitin ligase. TRIM28 can inhibit p53 through HDAC1-mediated deacetylation and direct ubiquitination in an MDM2-dependent manner. Thus, the overall impact of this study is high because of the identification of a novel mechanism in regulating wt-p53.

The other significant strengths of this manuscript included an apparent research strategy design and a clearly outlined and logically organized research approach. They provided both the in vitro and in vivo studies to evaluate the effect of PITAR. They offered reasonable control of the study by validating the results in cells with mutant p53. They also performed a rescue experiment to confirm the PITAR and TRIM28 relationship regulating p53. The conclusions were all supported by solid results. The overall data presentation is clear and convincing.

Author Response

Reviewer #1 (Public Review):

1. Only one PITAR siRNA was tested in majority of the experiments, which compromises the validity of the results. Some results are inconsistent. For example, Fig 2G indicates that PITAR siRNA caused G1 arrest. However, PITAR overexpression in the same cell line did not show any effect on cell cycle progression in Fig 5I.

We thank the reviewer for this comment. Indeed, we have used two siRNAs in experiments related to Fig. 2C, 2D, and 2E. Keeping the reviewer’s comment, we plan to reproduce the results of Fig. 2F, 2G, 2H, 2I, 5A, 5B, 5E, and supplementary Fig. 5A using additional siRNA targeting PITAR.

The reason for the fact that “PITAR silencing showed a robust G1 arrest, but PITAR overexpression failed to show any effect on the cell cycle profile” is as follows: since glioma cells overexpress PITAR (which keeps the p53 suppressed), silencing PITAR (which will elevate p53 levels) in glioma cells will show a robust phenotype in cell cycle profile (in the form of increase G1 arrest). In contrast, the overexpression of PITAR in glioma cells (which already has high levels of PITAR and hence drastically reduced p53 levels) is unlikely to show any significant change in the cell cycle profile. But, a phenotype for PITAR overexpression on cell cycle profile can be shown in DNA-damaged (which induces p53 levels) glioma cells. Indeed, we have done this experiment in Fig. 5L, which shows G2/M arrest (42.34%) induced by DNA damage is reduced significantly (19%) in PITAR overexpressed condition (34.42%). However, keeping reviewers' comments in the right spirit, we plan to repeat this experiment with appropriate modifications to arrive at a more robust phenotype for PITAR overexpression.

1. The conclusion that PITAR inactivates p53 through regulating TRIM28, which is highlighted in the title of the manuscript, is not supported by convincing results. Although the authors showed that a PITAR siRNA increased while PITAR overexpression decreased p53 level, the siRNA only marginally increased the stability of p53 (Fig 5E). The p53 ubiquitination level was barely affected by PITAR overexpression in Fig 5F. To convincingly demonstrate that PITAR regulates p53 through TRIM28, the authors need to show that this regulation is impaired/compromised in TRIM28-knockout conditions. The authors only showed that TRIM28 overexpression suppressed PITAR siRNAinduced increase of p53, which is not sufficient. Note that only one cell line was investigated in Fig 5.

To address this issue, we will overexpress PITAR in TRIM28 silenced cells to show the requirement of TRIM28 for PITAR to inhibit p53. In addition, we also plan to carry out PITAR silencing and overexpression experiments in another glioma cell line as recommended by the reviewer.

1. Another major weakness of this manuscript is that the authors did not provide any evidence indicating that the glioblastoma-promoting activities of PITAR were mediated by its regulation of p53 or TRIM28 (Fig 6 and Fig 7). Thus, the regulation of glioblastoma growth and the regulation of TRIM28/p53 appear to be disconnected.

We would like to respectfully disagree with the reviewer on this particular point. We have indeed provided the following evidence in the current version of the manuscript glioblastomapromoting activities of PITAR were mediated by its regulation of p53 or TRIM28.

A) In Fig. 6, we demonstrate that PITAR silencing-induced reduction in the neurosphere growth is accompanied by a reduction in TRIM28 RNA and an increase in the CDKN1A RNA without a change in p53 RNA levels. We also demonstrate that PITAR overexpression-induced neurosphere growth is accompanied by an increase in the TRIM28 RNA, and a decrease in CDKN1A RNA without a change in p53 RNA levels.

B) To add strength to the above results, we plan to do western blot experiments under similar conditions to demonstrate the appropriate changes in TRIM28, p53, and CDKN1A levels. Also, we will do a TRIM28 rescue experiment in RG5 neurosphere cells.

C) In supplementary Fig. 6 (related to Fig. 6), we show that PITAR silencing failed to decrease neurosphere growth in mutant p53 containing GSC line (MGG8).

D) In supplementary Fig. 7 (related to Fig. 6), we show that PITAR silencing failed to inhibit colony growth of p53-silenced U87 glioma cells (U87/shp53#1). We also show that while PITAR silencing decreased TRIM28 RNA levels in U87/shNT and U87/shp53#1 glioma cells, it failed to increase CDKN1A and MDM2 (p53 targets) at the RNA level.

E) In Fig. 7, we show that the TRIM28 protein level is drastically reduced in small tumors formed by U87/siPITAR cells.

F) In supplementary Fig. 8 (related to Fig. 7), we show that glioma tumor formed by U87/PITAR OE express high levels of TRIM28 protein but reduced levels of p21 protein.

G) We also plan to do additional experiments, as described below, to demonstrate that glioblastoma-promoting activities of PITAR are indeed mediated by its regulation of p53 or TRIM28. We will demonstrate the inability of PITAR overexpression to induce the growth of glioma-tumor initiated by TRIM28 silenced U87 cells.

1. It is not clear what kind of message the authors tried to deliver in Fig 7F/G. Based on the authors' hypothesis, DNA-damaging agents like TMZ would induce PITAR to inactivate p53, which would compromise TMZ's anti-cancer activity. However, the data show that TMZ was very effective in the inhibition of U87 growth. The authors may need to test whether PITAR downregulation, which would increase p53 activity, have any effects on TMZ-insensitive tumors. Such results are more therapeutically relevant.

Reviewer #1 rightly pointed out that TMZ induces PITAR expression, which should compromise TMZ's anti-cancer activity. In addition, overexpression of PITAR also promotes glioma-tumor growth. Figure 7F&G demonstrates the following two facts:1. PITAR overexpression increases the glioma-tumor growth (Figure 7G, compare red line with the blue line), 2. PITAR overexpressing glioma-tumor are resistant to TMZ chemotherapy (Figure 7G, compare the pink line with the green line).

In addition, in Figure 2I, we indeed show that PITAR-silenced cells are more sensitive to TMZ and Adriamycin chemotherapy.

However, considering reviewers’ comments, we plan to repeat Figure 7A, combining TMZ chemotherapy and PITAR silencing to demonstrate that TMZ chemotherapy-induced PITAR indeed promotes chemo-resistance.

1. Lastly, the model presented in Fig 7H is confusing. It is not clear what the exact role of PITAR in the DNA damage response based on this model. If DNA damage would induce PITAR expression, this would lead to inactivation of p53 as revealed by this manuscript. However, DNA damage is known to activate p53. Do the authors want to imply that PITAR induction by DNA damage would help to bring down the p53 level at the end of DNA damage response? The presented data do not support this role unfortunately.

We appreciate reviewer #1 comments. Based on our model in 7H, we believe DNA damageinduced PITAR attenuates DNA damage response by increasing TRIM28 protein levels. TRIM28 ubiquitinates p53 in an MDM2-dependent manner ( Wang et al., 2005). Based on this, we hypothesised that PITAR-induced TRIM28 also contributes to MDM2 mediated ending of DNA damage response.

Considering the reviewers' comments, we plan to do the following experiment.

The kinetics of p53, TRIM28, p21, MDM2 protein levels, and PITAR RNA levels after DNA damage will be monitored in PITAR-silenced conditions. It is known that reduction in the DNA damage-induced p53 levels coincides with high levels of MDM2 accumulation. We believe that in PITAR-silenced cells, p53 levels will remain high for a longer time compared to control cells because of the lack of PITAR-induced TRIM28-mediated degradation of p53.