No coin nor oath required. For personal study only.
Objective: to investigate the role of the stem cell regulator NUMB, and the differential splicing of NUMB isoforms, in the growth of PNETs. Methods: We investigated the expression of NUMB mRNA in tumor samples and cell lines derived from neuroblastoma, Ewing sarcoma, and medulloblastoma, using RT-PCR. We determined the expression and intracellular localization of NUMB protein in tumor samples using immunocytochemistry. We then transduced NUMB-GFP fusion constructs to study isoform-specific intracellular trafficking of NUMB protein.
Results: In all PNETs, we found novel NUMB splice variants lacking exon 8. This region falls between the 2 regions already known to be differentially spliced, phosphotyrosine binding (PTB) domain and proline rich region (PRR). Compared to developing brain, an embryonic tissue in which NUMB is actively transcribed, transcripts lacking exon 8 were highly enriched in PNETs and derivative cell lines. Immunocytochemistry confirmed that NUMB protein was expressed in many neuroectodermally derived tumors, with variable, tumor-specific patterns of intracellular localization: peripheral, cytoplasmic, asymmetric, and nuclear. We were able to replicate the diversity of intracellular localizations in vitro by transducing specific NUMB isoforms as GFP fusion proteins. Conclusions: NUMB, a key regulator of stem cell proliferation, is expressed in PNETs, and is spliced in patterns that are tumor specific. Splice variants of NUMB are localized differently within tumor cells, and suggesting isoform specific functions. Accordingly, tumor specific splicing of NUMB suggests a significant role of NUMB in governing PNET biology.
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