Abstract
Eyes Absents (EYA) are multifunctional proteins best known for their role in organogenesis. There is accumulating evidence that overexpression of EYAs in breast and ovarian cancers, and in malignant peripheral nerve sheath tumors, correlates with tumor growth and increased metastasis. The EYA protein is both a transcriptional activator and a tyrosine phosphatase, and the tyrosine phosphatase activity promotes single cell motility of mammary epithelial cells. Since EYAs are expressed in vascular endothelial cells and cell motility is a critical feature of angiogenesis we investigated the role of EYAs in this process. Using RNA interference techniques we show that EYA3 depletion in human umbilical vein endothelial cells inhibits transwell migration as well as Matrigel-induced tube formation. To specifically query the role of the EYA tyrosine phosphatase activity we employed a chemical biology approach. Through an experimental screen the uricosuric agents Benzbromarone and Benzarone were found to be potent EYA inhibitors, and Benzarone in particular exhibited selectivity towards EYA versus a representative classical protein tyrosine phosphatase, PTP1B. These compounds inhibit the motility of mammary epithelial cells over-expressing EYA2 as well as the motility of endothelial cells. Furthermore, they attenuate tubulogenesis in matrigel and sprouting angiogenesis in the ex vivo aortic ring assay in a dose-dependent fashion. The anti-angiogenic effect of the inhibitors was also demonstrated in vivo, as treatment of zebrafish embryos led to significant and dose-dependent defects in the developing vasculature. Taken together our results demonstrate that the EYA tyrosine phosphatase activity is pro-angiogenic and that Benzbromarone and Benzarone are attractive candidates for repurposing as drugs for the treatment of cancer metastasis, tumor angiogenesis, and vasculopathies.
Highlights
The Eyes Absent (EYA1-4) genes encode an unusual family of proteins; they have both transactivation and threonine phosphatase [1] activities in a poorly conserved N-terminal domain and tyrosine phosphatase activity in a well-conserved C-terminal domain [2,3,4]
Prior to investigating a cell-autonomous requirement of Eya in endothelial cells function, RT-PCR was performed in order to identify which Eya transcripts are expressed in vascular endothelial cells. mRNA for EYA1 and EYA3 was strongly detected in human umbilical vein endothelial cells (HUVECs) (Figure 1a)
To evaluate the role of Eyes Absents (EYA) in endothelial cell function HUVECs were stably infected with lentiviruses expressing either control shRNA or an shRNA specific to EYA3
Summary
The Eyes Absent (EYA1-4) genes encode an unusual family of proteins; they have both transactivation and threonine phosphatase [1] activities in a poorly conserved N-terminal domain and tyrosine phosphatase activity in a well-conserved C-terminal domain [2,3,4]. The threonine phosphatase activity has been linked with the innate immune response, while the tyrosine phosphatase activity is associated with cell motility [5], DNA damage repair [6,7], and fly eye development [2,3]. Achieving specificity among protein tyrosine phosphatases has been a challenge for inhibitor design. In this regard the EYAs could have an advantage since the EYA tyrosine phosphatase domain is mechanistically unusual; it does not utilize a cysteine residue in catalysis, as do all other known protein tyrosine phosphatases (PTPs) (reviewed in [21]). The active site of the EYAs represents an unconventional target for the design of small molecule PTP inhibitors
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
