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Investigations into the cellular pathways underlying ETV6-NTRK3-mediated transformation Lannon, Christopher L.

Abstract

Receptor tyrosine kinases are integral components of cellular signaling pathways, and are frequently deregulated in malignancies. There is increasing interest in the potential role of the NTRK family of neurotrophin receptors in human neoplasia. These proteins are known to mediate neuronal cell survival and differentiation, but altered NTRK signaling has also been implicated in oncogenesis. The ETV6-NTRK3 (EN) gene fusion results from a t(12; 15)(pl3; q25) translocation, and occurs in human pediatric spindle cell sarcomas and secretory breast carcinoma. EN fusion transcripts encode a chimeric protein tyrosine kinase, formed by the fusion ofthe SAM dimerization domain of ETV6 and the tyrosine kinase domain of NTRK3. The resultant EN fusion protein functions as a constitutively active protein tyrosine kinase with potent transforming activity. Further, EN-mediated transformation is associated with activation of the Ras-MAPK and PI3K-Akt pathways, increased levels of cyclin DI, and constitutive phosphorylation of the insulin receptor substrate 1 (IRS-1) through an interaction with the phosphotyrosine binding (PTB) domain of IRS-1. We have recently identified two C-terminal mutants (Δ614 and Y615F) that abolish and reduce binding to IRS-1, and subsequent transformation. Neither this region nor full-length EN protein contain any known PTB interaction motifs (i.e., NPXY); however, it does contain a TPIY, which may function as a putative PTB recognition sequence. TPIY mutants appear to abrogate anchorage-independent growth (soft agar assay and spheroid formation). These mutants most likely introduce a conformational change that affects protein interactions N-terminal to this region. However, these mutants appear to still bind IRS-1. Therefore, IRS-1 binding does not guarantee transformation in EN-expressing cells. To elucidate the role of EN in tumourigenesis, we next generated transgenic mice expressing ETV6-NTRK3 under the direction of two ubiquitously expressing promoters (CMV or β-actin/CMV), as this fusion protein appears to be implicated in a range of tumour types. We developed 13 independent founder mouse strains. Approximately 30% ofthe transgenic mice from two independent strains developed lymphomas after a long latency period. Additionally, a single mouse developed a fibrosarcoma expressing EN; this lesion was histologically identical to clinical cases of EN-expressing congenital fibrosarcoma. The low penetrance of tumour formation coupled with the advanced age of the mice raises several questions as to the cellular environment that may be prerequisite for ETV6-NTRK3 oncogenesis in these mice.

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