Stabilization of HIF-1α and HIF-2α, up-regulation of MYCC and accumulation of stabilized p53 constitute hallmarks of CNS-PNET animal model

Abstract

Recently, we described a new animal model of CNS primitive neuroectodermal tumors (CNS-PNET), which was generated by orthotopic transplantation of human Radial Glial (RG) cells into NOD-SCID mice’s brain sub-ventricular zone. In the current study we conducted comprehensive RNA-Seq analyses to gain insights on the mechanisms underlying tumorigenesis in this mouse model of CNS-PNET. Here we show that the RNA-Seq profiles derived from these tumors cluster with those reported for patients’ PNETs. Moreover, we found that (i) stabilization of HIF-1α and HIF-2α, which are involved in mediation of the hypoxic responses in the majority of cell types, (ii) up-regulation of MYCC, a key onco-protein whose dysregulation occurs in ~70% of human tumors, and (iii) accumulation of stabilized p53, which is commonly altered in human cancers, constitute hallmarks of our tumor model, and might represent the basis for CNS-PNET tumorigenesis in this model. We discuss the possibility that these three events might be interconnected. These results indicate that our model may prove invaluable to uncover the molecular events leading to MYCC and TP53 alterations, which would be of broader interest considering their relevance to many human malignancies. Lastly, this mouse model might prove useful for drug screening targeting MYCC and related members of its protein interaction network.

Characterization of brain tumor initiating cells isolated from an animal model of CNS primitive neuroectodermal tumors

“…In comparison to the RG self-renewing cells, the corresponding TCLs showed similar or increased expression of the RG marker BLBP, primitive neuroectodermal marker OTX2, RG-BTIC markers SOX2, Vimentin and Nestin, along with the BTIC marker OCT3/4 (Figure 2 ). Furthermore, similarly to the tumors generated by the RG cells [ 8 ], the TCL generated tumors also demonstrated high expression levels of these markers (Figure 1C1, 1C2, 1C4, 1D1-1D4 ). Moreover, as was the case for the TCL self-renewing cells, the TCL derived tumors exhibited high expression of onco-protein – cMyc (Figure 2 , Figure 3A1, 3A2 ).…”

“…Moreover, as was the case for the TCL self-renewing cells, the TCL derived tumors exhibited high expression of onco-protein – cMyc (Figure 2 , Figure 3A1, 3A2 ). Remarkably, the tumors generated by the RG cells [ 8 ], the TCLs and the TCL derived tumors exhibited aberrant accumulation of tumor-suppressor p53 (Figure 3B1-3B4, 3C1-3C4 ). Similarly, as the tumors generated by the RG cells [ 8 ], the TCL derived tumors displayed elevated level of p53 inhibitor - MDM2 (Figure 3D1-3D4 ), which might indicate p53 aberrant modification, rendering resistance to MDM2 mediated degradation [ 8 ].…”

“…These regulators may play a significant role in the BTIC maintenance [ 25 – 34 ]. It is conceivable that the up- regulation of these epigenetic regulators may be caused by the hypoxic microenvironment that is characteristic of the SVZ of the 3 rd ventricle, as we hypothesized in our previous study [ 8 ]. Such up-regulation might trigger the majority of the identified transcriptomic changes (Table 1 ), while enabling tumor formation in the motor cortex and cerebellum ( Supplementary Figure 1 ) - two locations where the injected RG cells did not show any sign of tumorigenic transformation [ 7 ].…”

“…Total RNA isolation was performed as mentioned above. cDNA synthesis and real-time quantitative reverse transcription-polymerase chain reactions (qRT-PCR) were performed as previously described [ 8 ]. QuantStudio 7 instrument (Applied Biosystems, USA) along with PowerUP SYBR Green Master Mix (A25742, Thermo Fisher Scientific, USA) were used according to the manufacturer’s instructions.…”

“…Recently, we described an animal model of CNS-PNET that was generated by orthotopic transplantation of human Radial Glial (RG) cells - the progenitor cells for adult NSCs - into NOD-SCID mice sub-ventricular zone of the brain [ 7 ], and proposed that BTICs may play a role in the maintenance of these tumors [ 8 ]. We documented expression of RG-BTIC markers such as SOX2, Vimentin and Nestin, BTIC marker OCT3/4, up-regulation of onco-protein cMyc, along with an aberrant accumulation of stabilized tumor-suppressor protein p53 in the model tumors [ 8 ].…”