Biology subjects

Stapleton, S.

Publications and source records attributed to Stapleton, S..

3 recordsLinked to original sources

bioRxiv2024

Unravelling the Impact of miR-21 Overexpression on the MicroRNA Network and Cancer Pathways

AbstractMicroRNAs (miRNA, miRs) are small noncoding RNAs that are ubiquitously expressed in all mammalian cells. Their primary function is the regulation of nascent RNA transcripts by direct binding to regions on the target. There is now exciting data to suggest that these miRNAs can bind to other miRNAs, and this may have a broader impact on gene regulation in disease states. The oncomiR miR-21 is one of the highest-expressing miRNAs in cancer cells, and in this study, we characterise which miRNAs could be potential targets of miR-21. In cancer cells delivered with a miR-21 mimic, there was an observable shift of the miRNA milieu. We demonstrate that the miR-17-92a cluster, which harbours six miRNA members, may be a target of miR-21 regulation. Additionally, the primary transcript of miR-17-92a was reduced in the presence of miR-21. In the broader context of gene regulation, overexpression of miR-21 shifted the expression of more than 150 miRNAs, including those known to regulate genes encoding proteins in cancer pathways such as the MAPK signalling and FoxO pathways. This study expands upon our limited understanding of miR:miR regulatory network and reinforces the concept that miRNAs can regulate each other, thereby influencing broader gene regulatory networks.

cell biology↗

bioRxiv2020

Drug screening linked to molecular profiling identifies novel dependencies in patient-derived primary cultures of paediatric high grade glioma and DIPG

Paediatric high grade glioma and diffuse midline glioma (including DIPG) are comprised of multiple biological and clinical subgroups, the majority of which urgently require novel therapies. Patient-derived in vitro primary cell cultures represent potentially useful tools for mechanistic and preclinical investigation based upon their retention of key features of tumour subgroups under experimental conditions amenable to high-throughput approaches. We present 17 novel primary cultures derived from patients in London, Dublin and Belfast, and together with cultures established or shared from Barcelona, Brisbane, Rome and Stanford, assembled a panel of 52 models under 2D (laminin matrix) and/or 3D (neurospheres) conditions, fully credentialed by phenotypic and molecular comparison to the original tumour sample (methylation BeadArray, panel/exome sequencing, RNAseq). In screening a subset of these against a panel of ~400 approved chemotherapeutics and small molecules, we identified specific dependencies associated with tumour subgroups and/or specific molecular markers. These included MYCN-amplified cells and ATM/DNA-PK inhibitors, and DIPGs with PPM1D activating truncating mutations and inhibitors of MDM2 or PARP1. Specific mutations in PDGFRA were found to confer sensitivity to a range of RTK inhibitors, though not all such mutations conferred sensitivity to targeted agents. Notably, dual PDGFRA/FGFR and downstream pathway MEK inhibitors showed profound effects against both PDGFRA-sensitising mutant and FGFR1-dependent non-brainstem pHGG and DIPG. In total, 85% cells were found to have at least one drug screening hit in short term assays linked to the underlying biology of the patients tumour, providing a rational approach for individualised clinical translation.

cancer biology↗

bioRxiv2020

The long non-coding RNA lnc-HLX-2-7 is oncogenic in group 3 medulloblastomas

BackgroundMedulloblastoma (MB) is an aggressive brain tumor that predominantly affects children. Recent high-throughput sequencing studies suggest that the non-coding RNA genome, in particular long non-coding RNAs (lncRNAs), contributes to MB sub-grouping. Here we report the identification of a novel lncRNA, lnc-HLX-2-7, as a potential molecular marker and therapeutic target in group 3 MBs. MethodsPublicly available RNA sequencing (RNA-seq) data from 175 MB patients were interrogated to identify lncRNAs that differentiate between MB subgroups. After characterizing a subset of differentially expressed lncRNAs in vitro and in vivo, the group 3-enriched lncRNA lnc-HLX2-7 was deleted by CRISPR/Cas9 in the MB cell line D425 Med. Intracranially injected tumors were further characterized by bulk and single-cell RNA-sequencing. Resultslnc-HLX-2-7 is highly upregulated in group 3 MB cell lines, patient-derived xenografts, and primary MBs compared to other MB sub-groups as assessed by qRT-PCR, RNA-seq, and RNA fluorescence in situ hybridization (FISH). Depletion of lnc-HLX-2-7 with antisense oligonucleotides or CRISPR/Cas9 significantly reduced cell proliferation and 3D colony formation and induced apoptosis. lnc-HLX-2-7-deleted D425 Med cells injected into mouse cerebella produced smaller tumors than those derived from parental cells. Pathway analysis revealed that lnc-HLX2-7 modulated oxidative phosphorylation, mitochondrial dysfunction, and sirtuin signaling pathways. The MYC oncogene regulated lnc-HLX-2-7, and the small molecule BET-bromodomain (BRD4) inhibitor JQ1 reduced lnc-HLX2-7 expression. Conclusionslnc-HLX-2-7 is oncogenic in MB and represents a promising novel molecular marker and a potential therapeutic target in group 3 MBs in children. Key pointsO_LIlnc-HLX-2-7 is highly upregulated in group 3 medulloblastomas compared to other sub-groups. C_LIO_LIIn vitro and in vivo studies strongly support an oncogenic role for lnc-HLX2-7 in group 3 medulloblastoma. C_LIO_LIlnc-HLX-2-7 may be a novel biomarker and a potential therapeutic target in group 3 medulloblastoma. C_LI Importance of the studyGroup 3 medulloblastomas are associated with poor clinical outcomes, are difficult to subtype clinically, and their biology is poorly understood. In an effort to address these problems, we identified a group 3-specific long non-coding RNA, lnc-HLX-2-7, in an in silico analysis of 175 medulloblastomas and confirmed its expression in group 3 medulloblastoma cell lines, patient-derived xenografts, and FFPE samples. CRISPR/Cas9 deletion and antisense oligonucleotide knockdown of lnc-HLX-2-7 significantly reduced cell growth and 3D colony formation and induced apoptosis. Deletion of lnc-HLX-2-7 in cells injected into mouse cerebellums reduced tumor growth compared to parental cells, and RNA sequencing of these tumors revealed lnc-HLX-2-7-associated modulation of cell viability and cell death signaling pathways. The oncogene MYC regulates lnc-HLX-2-7, and its expression can be controlled by the BET-bromodomain (BRD4) inhibitor JQ1. lnc-HLX-2-7 is a candidate biomarker and a potential therapeutic target in group 3 medulloblastomas in children.

cancer biology↗