Abstract
Background
SOX2 is a transcription factor associated with stem cell activity in several tissues. SOX2 expression is elevated in a large proportion of cancers, such as glioblastoma (GB), the most common malignant primary brain tumor in adults. SOX2 acts as a relevant driver of GB progression and high levels are associated with the population of tumor initiating cells and poor patient outcome. Transcriptional regulation of SOX2 is complex and not fully understood. The SOX2 regulatory region 2 (SRR2) is located downstream of the SOX2 coding region and mediates SOX2 expression by the association of p21CIP1 and p27KIP1 to SRR2. In this study we dissected the role of SRR2 on SOX2 activity in GB.
Material and Methods
We deleted
SRR2 (SRR2del) by CRISPR/Cas9 technology in U373 GB cells. We analyzed the expression of SOX2 and performed
in vitro functional experiments. We accomplished rescue experiments overexpressing SOX2 in
SRR2del cells and we studied the link between SOX2, p21
CIP1 and p27
KIP1. Finally, we carried out an
in vivo carcinogenesis assay by the injection of
SRR2del cells in immunodeficient mice.
Results
We report that the deletion of
SRR2 in GB cells leads to a reduction of SOX2 expression, which was accompanied with an impairment of cell growth and proliferation as well as with a reduction of self-renewal capacity
in vitro. These data reveal that
SRR2 is required for SOX2 expression and that its deletion results in impaired tumorigenic capacity of GB cells. These effects correlated with an increased expression of p21
CIP1 and p27
KIP1 together with high levels of p53 in
SRR2del cells. On the contrary,
SRR2del cells presented decreased levels of stem cell markers, supporting the idea that
SRR2 is necessary for the SOX2-mediated regulation of stemness pathways. Furthermore, ectopic overexpression of SOX2 in
SRR2del cells restored SOX2 expression as well as proliferation and stem cell properties, indicating that the expression and the oncogenic activity of SOX2 is regulated by
SRR2. Additionally, our xenograft models re vealed that the lack of
SRR2 impairs tumor initiation and growth
in vivo. Tumors derived from
SRR2del cells were significantly smaller compared to controls and exhibit low levels of SOX2, Ki67 and BMI1 but high levels of p21
CIP1 and p27
KIP1.
Conclusion
Our data confirm that the
SRR2 regulates SOX2 expression and reveal that
SRR2 deletion reduces SOX2 levels and halts malignant activity driven by SOX2 in GB.
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