Aneurysmal bone cyst (ABC) is an aggressive, pediatric bone tumor characterized by extensive destruction of the surrounding bone. Though first described over 60 years ago, its molecular etiology remains poorly understood. Recent work revealed that ABCs harbor translocation of TRE17/USP6, leading to its transcriptional upregulation. TRE17 encodes a ubiquitin-specific protease (USP), and a TBC domain that mediates binding to the Arf6 GTPase. However, the mechanisms by which TRE17 overexpression contributes to tumor pathogenesis, and the role of its USP and TBC domains are unknown. ABCs are characterized by osteolysis, inflammatory recruitment, and extensive vascularization, processes in which matrix proteases play a prominent role. This led us to explore whether TRE17 regulates the production of matrix metalloproteinases (MMPs). In the current study, we demonstrate that TRE17 is sufficient to induce expression of MMP-9 and MMP-10, in a manner requiring its USP activity, but not its ability to bind Arf6. TRE17 induces transcription of MMP-9 through activation of NFκB, mediated in part by the GTPase RhoA and its effector kinase, ROCK. Furthermore, xenograft studies demonstrate that TRE17 induces formation of tumors that reproduce multiple features of ABC, including a high degree of vascularization, with an essential role for the USP domain. In sum, these studies reveal that TRE17 is sufficient to initiate tumorigenesis, identify MMPs as novel TRE17 effectors that likely contribute to ABC pathogenesis, and define the underlying signaling mechanism of their induction.

Overexpression and/or activity of c-Src non-receptor tyrosine kinase is associated with progression of several human epithelial cancers including breast cancer. c-Src activity in 'pure' ductal carcinoma in situ (DCIS) was measured to assess whether this predicts recurrence and/or correlates with HER2 expression and other clinical parameters. Activated c-Src levels were evaluated in DCIS biopsies from 129 women, with median follow-up at 60 months. High levels of activated c-Src correlated with HER2 positivity, high tumour grade, comedo necrosis and elevated epithelial proliferation. In univariate analysis, high activated c-Src level associated with lower recurrence-free survival at 5 years (P ¼ 0.011). Thus, high c-Src activity may identify a subset of DCIS with high risk of recurrence or progression to invasive cancer where therapeutics targeting c-Src may benefit this patient subset.

The C-terminal hypervariable domain of K-Ras4B targets the protein to the plasma membrane by a combination of positive charge and a hydrophobic signal (farnesyl group). We analysed the contribution of several structural features of the domain: net charge, charge distribution, amino acid sequence and lipid speci®city to membrane targetting and function by using arti®cial hypervariable' domains fused to either EGFP or V12K-Ras4B. We found that charge and a lipid residue are sucient for plasma membrane localization and function of the constitutively active V12K-Ras4B. However, the amount of net charge, charge distribution and the length of the anchoring domain are important. Increasing the net charge and concentrating it close to the C-terminus increases not only the percentage of membrane bound protein, but also shifts the distribution from internal membranes, including the nuclear envelope, to the plasma membrane. While plasma membrane binding is necessary for V12K-Ras4B activity (MAPK activation and focus formation), we found that there are additional restrictions. In particular, mutants with very highly charged domains that bind almost exclusively to the plasma membrane show less transforming potential than expected. In addition, a construct with a short`hypervariable' domain (7 amino acids) also has decreased transformation activity. These results suggest that speci®c interactions between K-Ras4B and the plasma membrane are required. Oncogene (2000) 19, 4582 ± 4591.

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We report the development and application of photoactivatable Green Cherry (G PA C), the first genetically encoded "continuously red-photoactivatable green" two-color probe for live cell imaging. G PA C is unique in that it enables real-time tracking of selected subpopulations of proteins and organelles in the cell or of cells within tissues and whole organisms, with constant reference to the entire population of the probe. Using G PA C-zyxin as proof of utility, we obtained new insights into the dynamic movement of the cytoskeletal protein zyxin. We show that zyxin is continuously and rapidly recruited from the cytosol into established focal adhesions. It can also move rapidly within a given focal adhesion and "hop" between adjacent focal adhesions, emphasizing the dynamic nature of proteins within these structures. The in vivo utility of G PA C is exemplified by tracking hemocyte movements using a versatile transgenic Drosophila model engineered to express G PA C in tissues and cells of interest under the control of the GAL4-inducible promoter.