Recently, messenger RNAs in eukaryotes have shown to associate with antisense (AS) transcript partners that are often referred to as long noncoding RNAs (lncRNAs) whose function is largely unknown. Here, we have identified a natural AS transcript for tyrosine kinase containing immunoglobulin and epidermal growth factor homology domain-1 (tie-1), tie-1AS lncRNA in zebrafish, mouse, and humans. In embryonic zebrafish, tie-1AS lncRNA transcript is expressed temporally and spatially in vivo with its native target, the tie-1 coding transcript and in additional locations (ear and brain). The tie-1AS lncRNA selectively binds tie-1 mRNA in vivo and regulates tie-1 transcript levels, resulting in specific defects in endothelial cell contact junctions in vivo and in vitro. The ratio of tie-1 versus tie-1AS lncRNA is altered in human vascular anomaly samples. These results directly implicate noncoding RNA-mediated transcriptional regulation of gene expression as a fundamental control mechanism for physiologic processes, such as vascular development.

Brain metastasis is a defining component of tumor pathophysiology, and the underlying mechanisms responsible for this phenomenon are not well understood. Current dogma is that tumor cells stimulate and activate astrocytes, and this mutual relationship is critical for tumor cell sustenance in the brain. Here, we provide evidence that primary rat neonatal and adult astrocytes secrete factors that proactively induced human lung and breast tumor cell invasion and metastasis capabilities. Among which, tumor invasion factors namely matrix metalloprotease-2 (MMP-2) and MMP-9 were partly responsible for the astrocyte media-induced tumor cell invasion. Inhibiting MMPs reduced the ability of tumor cell to migrate and invade in vitro. Further, injection of astrocyte media-conditioned breast cancer cells in mice showed increased invasive activity to the brain and other distant sites. More importantly, blocking the preconditioned tumor cells with broad spectrum MMP inhibitor decreased the invasion and metastasis of the tumor cells, in particular to the brain in vivo. Collectively, our data implicate astrocyte-derived MMP-2 and MMP-9 as critical players that facilitate tumor cell migration and invasion leading to brain metastasis.

Metastatic outcomes depend on the interactions of metastatic cells with a specific organ microenvironment. Our previous studies have shown that triple-negative breast cancer (TNBC) MDA-MB-231 cells passaged in astrocyte-conditioned medium (ACM) show proclivity to form brain metastases, but the underlying mechanism is unknown. The combination of microarray analysis, qPCR, and ELISA assay were carried out to demonstrate the ACM-induced expression of angiopoietin-like 4 (ANGPTL4) in TNBC cells. A stable ANGPTL4-knockdown MDA-MB-231 cell line was generated by ANGPTL4 short-hairpin RNA (shRNA) and inoculated into mice via left ventricular injection to evaluate the role of ANGPTL4 in brain metastasis formation. The approaches of siRNA, neutralizing antibodies, inhibitors, and immunoprecipitation were used to demonstrate the involved signaling molecules. We first found that ACM-conditioned TNBC cells upregulated the expression of ANGPTL4, a secreted glycoprotein whose effect on tumor progression is known to be tumor microenvironment- and tumor-type dependent. Knockdown of ANGPTL4 in TNBC MDA-MB-231 cells with shRNA decreased ACM-induced tumor cell metastatic growth in the brain and attributed to survival in a mouse model. Furthermore, we identified that astrocytes produced transforming growth factor-beta 2 (TGF-β2), which in part is responsible for upregulation of ANGPTL4 expression in TNBC through induction of SMAD signaling. Moreover, we identified that tumor cells communicate with astrocytes, where tumor cell-derived interleukin-1 beta (IL-1β) and tumor necrosis factor alpha (TNF-α) increased the expression of TGF-β2 in astrocytes. Collectively, these findings indicate that the invading TNBC cells interact with astrocytes in the brain microenvironment that facilitates brain metastases of TNBC cells through a TGF-β2/ANGPTL4 axis. This provides groundwork to target ANGPTL4 as a treatment for breast cancer brain metastases.

Mitogen-activated protein kinases play an integral role in several cellular processes. To regulate mitogen-activated protein kinases, cells express members of a counteracting group of proteins called phosphatases. In this study, we have identified a specific role that one member of this family of phosphatases, dual-specific phosphatase-5 (Dusp-5) plays in vascular development in vivo. We have determined that dusp-5 is expressed in angioblasts and in established vasculature and that it counteracts the function of a serine threonine kinase, Snrk-1, which also plays a functional role in angioblast development. Together, Dusp-5 and Snrk-1 control angioblast populations in the lateral plate mesoderm with Dusp-5 functioning downstream of Snrk-1. Importantly, mutations in dusp-5 and snrk-1 have been identified in affected tissues of patients with vascular anomalies, implicating the Snrk-1–Dusp-5 signaling pathway in human disease.

The stringent expression of the hypoxia inducible factor-1α (HIF-1α) is critical to a variety of pathophysiological conditions. We reveal that, in normoxia, enzymatic action of cystathionine β-synthase (CBS) produces H₂S, which persulfidates prolyl hydroxylase 2 (PHD2) at residues Cys²¹ and Cys³³ (zinc finger motif), augmenting prolyl hydroxylase activity. Depleting endogenous H₂S either by hypoxia or by inhibiting CBS via chemical or genetic means reduces persulfidation of PHD2 and inhibits activity, preventing hydroxylation of HIF-1α, resulting in stabilization. Our in vitro findings are further supported by the depletion of CBS in the zebrafish model that exhibits axis defects and abnormal intersegmental vessels. Exogenous H₂S supplementation rescues both in vitro and in vivo phenotypes. We have identified the persulfidated residues and defined their functional significance in regulating the activity of PHD2 via point mutations. Thus, the CBS/H₂S/PHD2 axis may provide therapeutic opportunities for pathologies associated with HIF-1α dysregulation in chronic diseases.

Rho GTPases play an important and versatile role in several biological processes. In this study, we identified the zebrafish ortholog of the mammalian Rho A guanine exchange factor, synectin-binding guanine exchange factor (Syx), and determined its in vivo function in the zebrafish and the mouse. We found that Syx is expressed specifically in the vasculature of these organisms. Loss-of-function studies in the zebrafish and mouse point to a specific role for Syx in angiogenic sprouting in the developing vascular bed. Importantly, vasculogenesis and angioblast differentiation steps were unaffected in syx knockdown zebrafish embryos, and the vascular sprouting defects were partially rescued by the mouse ortholog. Syx knockdown in vitro impairs vascular endothelial growth factor-A–induced endothelial cell migration and angiogenesis. We have also uncovered a potential mechanism of endothelial sprout guidance in which angiomotin, a component of endothelial cell junctions, plays an additive role with Syx in directing endothelial sprouts. These results identify Syx as an essential contributor to angiogenesis in vivo.