TRE17/ubiquitin-specific protease 6 (USP6) oncogene translocated in aneurysmal bone cyst blocks osteoblastic maturation via an autocrine mechanism involving bone morphogenetic protein dysregulation.

Aneurysmal bone cyst (ABC) is a pediatric osseous tumor characterized by extensive destruction of the surrounding bone. The molecular mechanisms underlying its pathogenesis are completely unknown. Recent work showed that translocation of the TRE17/USP6 locus occurs in over 60% of ABC cases resulting in TRE17 overexpression. Immature osteoblasts are presumed to be the cell type harboring translocation of TRE17 in at least a subset of ABCs. However, the effects of TRE17 overexpression on transformation and osteoblast function are unknown. TRE17 encodes a ubiquitin-specific protease (USP) and a TBC (TRE2-Bub2-Cdc16) domain that promotes activation of the Arf6 GTPase. Here we report that TRE17 potently inhibits the maturation of MC3T3 pre-osteoblasts in a USP-dependent and Arf6-independent manner. Notably, we find that TRE17 function is mediated through an autocrine mechanism. Transcriptome analysis of TRE17-expressing cells reveals dysregulation of several pathways with established roles in osteoblast maturation. In particular, signaling through the bone morphogenetic protein (BMP) pathway, a key regulator of osteogenesis, is profoundly altered. TRE17 simultaneously inhibits the expression of BMP-4 while augmenting the BMP antagonist, Gremlin-1. Osteoblastic maturation is restored in TRE17-expressing cells by the addition of exogenous BMP-4, thus establishing a functional role for BMP-4 during TRE17-induced transformation. Because bone homeostasis involves a precise balance between the activities of osteoblasts and osteoclasts, our studies raise the possibility that attenuated osteoblast maturation caused by TRE17 overexpression may contribute to the bone loss/destruction observed in ABC.

The alternatively-included 11a sequence modifies the effects of Mena on actin cytoskeletal organization and cell behavior.

During tumor progression, alternative splicing gives rise to different Mena protein isoforms. We analyzed how Mena11a, an isoform enriched in epithelia and epithelial-like cells, affects Mena-dependent regulation of actin dynamics and cell behavior. While other Mena isoforms promote actin polymerization and drive membrane protrusion, we find that Mena11a decreases actin polymerization and growth factor-stimulated membrane protrusion at lamellipodia. Ectopic Mena11a expression slows mesenchymal-like cell motility, while isoform-specific depletion of endogenous Mena11a in epithelial-like tumor cells perturbs cell:cell junctions and increases membrane protrusion and overall cell motility. Mena11a can dampen membrane protrusion and reduce actin polymerization in the absence of other Mena isoforms, indicating that it is not simply an inactive Mena isoform. We identify a phosphorylation site within 11a that is required for some Mena11a-specific functions. RNA-seq data analysis from patient cohorts demonstrates that the difference between mRNAs encoding constitutive Mena sequences and those containing the 11a exon correlates with metastasis in colorectal cancer, suggesting that 11a exon exclusion contributes to invasive phenotypes and leads to poor clinical outcomes.

Selectivity in subunit composition of Ena/VASP tetramers.

The members of the actin regulatory family of Ena/VASP proteins form stable tetramers. The vertebrate members of the Ena/VASP family, VASP, Mena and EVL, have many overlapping properties and expression patterns, but functional and regulatory differences between paralogues have been observed. The formation of mixed oligomers may serve a regulatory role to refine Ena/VASP activity. While it has been assumed that family members can form mixed oligomers, this possibility has not been investigated systematically. Using cells expressing controlled combinations of VASP, Mena and EVL, we evaluated the composition of Ena/VASP oligomers and found that VASP forms oligomers without apparent bias with itself, Mena or EVL. However, Mena and EVL showed only weak hetero-oligomerization, suggesting specificity in the association of Ena/VASP family members. Co-expression of VASP increased the ability of Mena and EVL to form mixed oligomers. Additionally, we found that the tetramerization domain (TD) at the C-termini of Ena/VASP proteins conferred the observed selectivity. Finally, we demonstrate that replacement of the TD with a synthetic tetramerizing coiled coil sequence supports homo-oligomerization and normal VASP subcellular localization.