The super-enhancer driven lncRNA PRKCQ-AS1 promotes neuroblastoma tumorigenesis

Abstract

While MYCN-amplified neuroblastoma has been the focus of neuroblastoma research in the past three decades, most human neuroblastomas do not harbour MYCN oncogene amplification, and their tumorigenic factors are unknown, highlighting the importance of identifying other oncogenic factors. Long noncoding RNAs (lncRNAs) play important roles in cancer oncogenesis. Here, I have shown the lncRNA PRKCQ-AS1 is upregulated by super-enhancers upstream of the PRKCQ-AS1 gene that drive its over-expression only in MYCN non-amplified neuroblastoma cell lines through the well-known super-enhancer components CDK7 and BRD4. Targeting CDK7 with THZ1 or targeting BRD4 with AZD5153 significantly downregulate the expression of the lncRNA PRKCQ-AS1, but not its neighbouring protein-coding gene PRKCQ or the well-documented super-enhancer[1]associated c-Myc oncogene. I have shown that PRKCQ-AS1 was a cytoplasmic lncRNA and bound to the RNA binding protein (RBP) MSI2 in MYCN non-amplified neuroblastoma and that their binding could be blocked by the MSI2 RBP inhibitor Ro 08–2750. I have also shown that MSI2 bound to two fragments of PRKCQ-AS1 RNA spanning about 300bp towards to the 5’ end of PRKCQ[1]AS1. Additionally, RNA immunoprecipitation and subsequent sequencing identified the oncogene BMX as the downstream target most significantly modulated by the PRKCQ-AS1 RNA and MSI2 protein interaction, which in turn activated the MEK/ERK signalling pathway. Functional studies revealed that PRKCQ-AS1 promoted MYCN-non-amplified neuroblastoma cell proliferation in vitro and tumour progression in vivo. The PRKCQ-AS1- binding protein MSI2 and their target BMX also promoted MYCN-non-amplified neuroblastoma cell proliferation. In human neuroblastoma tissues, there was a strong correlation between the expression of PRKCQ-AS1 and MSI2 and the expression of BMX, and high PRKCQ-AS1, MSI2 and BMX expression in human neuroblastoma tissues correlated with poor clinical outcome in patients. Additionally, high levels of both PRKCQ[1]AS1 and MSI2 expression were strong prognostic factors for poor patient outcome independent of the current standard markers including age at diagnosis, disease stage and MYCN gene amplification. My study therefore identified PRKCQ-AS1 and its binding protein MSI2 as factors important for MYCN-non-amplified neuroblastoma cell proliferation and tumorigenesis and the interaction between PRKCQ-AS1 and MSI2 as a valid target for the treatment of MYCN-non-amplified neuroblastoma.